In forensic anthropology, age estimation from adult skeletal remains is one of the field’s harder problems. Classical methods lose accuracy as people get older, and biological fluids — the tissue base for most methylation-based age estimation research — aren’t always what a scene gives you.
Maria Josefina Castagnola, a PhD student at New Jersey Institute of Technology, is working on an alternative. Her research focuses on DNA methylation in teeth — specifically dentin and pulp — as a tool for molecular age estimation. Teeth are chemically resilient, protected from environmental insults by their low porosity, and recoverable in contexts where soft tissue is long gone. That combination makes them worth studying carefully.
Josefina presented her poster at ISHI 36 as a Student Ambassador, joining the conference from Argentina by way of NJIT. We spoke with her about the research, the challenges of working at the edge of a still-developing protocol, and what brought her to this work in the first place.
Tell us about your research. What problem are you trying to solve, and what have you found so far?
The core challenge in forensic anthropology is age estimation in adults. Classical skeletal methods — examining bone wear, joint degeneration — become less reliable as people age, especially past 50. Forensic epigenetics has shown real promise as a molecular alternative, but most of the existing work uses biological fluids. Josefina’s lab, under her PI Dr. Sara Zapico, is building the case for skeletal tissues instead.
Her study uses dentin and pulp from extracted teeth, donated through a dentist’s office and from skeletal collections at Mercyhurst University. The work zeroes in on two questions: how little DNA can you start with and still get reliable methylation data, and what combination of markers and regression models gives the best age prediction.
The results so far are practical. Josefina found that 100 nanograms of starting DNA — half the optimized amount of 200 ng — produced no significant difference in methylation profiles. That matters for casework, where sample input is often limited by what the specimen can provide. On the modeling side, splitting the regression — running one model for the full lifespan and a separate one for individuals under 50 — improved prediction accuracy for younger individuals, though building a reliable model for the entire age range remains an open goal.
The marker ELOVL2 performed consistently, in line with what’s been reported in other tissues and fluids. Other markers from the published skeletal literature were less informative in dentin, which Josefina flags directly: the field is still working from markers discovered in other tissue types, and identifying dentin-specific CpG sites is one of the field’s cleaner next problems.
Why this topic, and why does it matter to you personally?
Josefina is direct about what draws her to this area. Contributing to human identification in mass disaster investigations and human rights cases is not abstract for her — it’s the stated goal. Forensic epigenetics is still developing its tools, and she wants to be part of making those tools usable in the contexts that need them most.
“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.”
That orientation is visible in how she talks about the research. The goal isn’t to optimize for accuracy metrics in the abstract — it’s to build something that reaches the communities and investigations that depend on these identifications.
What was unexpectedly challenging about the process?
Bisulfite conversion — the chemical step that allows methylated and unmethylated cytosines to be distinguished — degrades DNA. That’s a known limitation, but it becomes especially consequential when you’re already working with limited input from skeletal material. Getting complete, high-quality methylation profiles required careful optimization at every stage: tissue separation, fragmentation, extraction, quantification, conversion, amplification, sequencing.
Josefina was also new to pyrosequencing when this work began, learning the instrument under Dr. Zapico’s guidance while trying to standardize a protocol at the same time.
“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.”
What moments defined the research for you?
Two findings shaped the direction of the work. One was establishing that reliable methylation profiles could be obtained with 100 nanograms of DNA, opening the door to working with lower-input samples in the future. The other was discovering that several markers selected from the skeletal tissue literature weren’t informative in dentin — a negative result that clarified where the protocol needed to go next.
“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.”
That kind of recalibration — finding out what doesn’t work, and letting it redirect the study — is exactly what early-stage protocol development looks like.
What do you hope people take away from your poster?
Josefina’s answer is specific. Not a general endorsement of epigenetics, but a pointed claim about what this tissue type can contribute.
“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’s next for this research?
The immediate next steps are incremental and grounded: expanding the sample set, continuing to optimize the model, and identifying methylation markers specific to dentin and bone rather than adapting markers discovered in blood or other fluids. Further out, she’s interested in what happens to methylation data when remains have been exposed to fire or other environmental extremes — the conditions most likely to complicate recovery in the cases this work is ultimately meant to support.
“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.”
Josefina presented this work at ISHI 36 in West Palm Beach. For a field still developing the foundational protocols for skeletal tissue methylation, her early-stage results — and the clarity with which she frames what’s still unresolved — reflect exactly the kind of work the Student Ambassador program exists to surface.