Age estimation using DNA methylation from forensically relevant traces is a highly researched application in Forensic DNA Phenotyping at the moment. As it is currently being implemented in forensic casework in an increasing number of laboratories across the world, it is crucial to rigorously test the reliability and reproducibility of the various available models. Different tissue types require specific markers and models to achieve optimal age estimation accuracy. Bone tissue, in particular, has received less attention in DNA methylation analyses compared to other forensically relevant samples types like blood or saliva. Although markers and models for bones have been explored using different technologies (e.g., MPS or SNaPshot), the datasets are often small, and sample collection methods are not always well documented.
In ISHI Student Ambassador, Charlotte Sutter’s, study, she and her colleagues tested an existing MPS-based age estimation tool for bones which investigates the markers ELOVL2, KLF14, MIR29B2C/C1orf132, TRIM59, EDARADD, ASPA, FHL2 and PDE4C.
We chatted with Charlotte to learn a little more, and if you’ll be at ISHI this September, be sure to stop by during the poster sessions to learn more!
Briefly describe your work/area of interest.
Forensic age estimation based on methylation pattern changes in age-dependent CpG sites has gained increasing attention in recent years. When an STR profile from a stain found at a crime scene does not produce a hit in a DNA database, estimating a person’s age can help narrow down the stain donor. Since methylation patterns are highly tissue-specific, different target sites and statistical models are required to achieve the highest possible estimation accuracy. One of the forensically relevant tissues that has not been studied as intensively in methylation studies as others is bone tissue.
In my current project, I am comparing the performance of a published age estimation tool for bones on different bone types. We hypothesize that bone types like petrous bone, femur, clavicle, rib, or iliac crest might display different methylation patterns, resulting in differences in the estimated age. We are collecting these bone types from deceased individuals during autopsies and extract DNA from drilled bone powder. Afterwards, we sequence target amplicons on a MiSeq instrument and calculate the age from the methylation percentages at six target CpG sites. Finally, we correlate the age estimates with the chronological age at the time of death and investigate the methylation patterns in the amplicons in more detail.
This project would not be possible without my supervisors Prof. Dr. Cordula Haas and Dr. Jacqueline Neubauer as well as the team from the Institute of Pathology and Molecular Pathology from the University Hospital Zurich who provide us with all bone samples we need. In addition, much of the technical knowledge I need for this project comes from my time at Walther Parson’s laboratory for which I am very grateful. Finally, days in the lab drilling bones could be rather exhausting if it weren’t for my fellow students who cheer me up when I need it.
So far, we have collected bones from almost 20 individuals. It appears that the methylation patterns do not differ significantly between bone types, and the age estimates are overall comparable across the different bone types. We might find indications that the estimates from one of the five investigated types are slightly different from the others. However, as this is an ongoing project, only time (and more samples) will tell whether this statement holds true.
How did you become interested in this work? Why did this particular project appeal?
I have always found bones to be a particularly fascinating type of sample because they are, in my eyes, quite different from other “classical” forensic samples like blood, saliva, or sperm. Before my PhD, I regretted never having had the chance to work on bones, so when this project was proposed to me, I was immediately thrilled. Comparing different bone types for different methylation profiles seemed like a relevant research question that could contribute valuable information to the forensic scientific community.
What has been the most rewarding part of working on this project?
The most rewarding part of working on this project was when I first realized I managed to get good quality DNA from the bones. Bones are generally difficult to work with in terms of DNA quality and quantity, so I was really happy when I saw that the way I cleaned and drilled the bones resulted in good quality, non-contaminated DNA extracts. Although the bones I am using are fresh and certainly not as difficult to handle as older bones, I am still relieved every time I get a nice STR profile in the end.
Are there any further areas of research you think are needed based on your findings?
We definitely need more research on older bones, which we also strive to do later in this project. Although it is certainly important to know more about methylation patterns in fresh bone samples in general, forensic scenarios will likely involve older, maybe decomposed and environmentally exposed bones whose methylation profiles might have been changed. In addition, if it holds true that not all bone types display similar methylation patterns, an adjustment or a remodeling of existing age estimation tools for bones might be needed depending on the investigated bone type.
How do you hope your research will be applied in real-world scenarios?
When talking about bones in forensic scenarios, we mostly refer to the identification of remains. I hope that my research might contribute to identifying remains whose chronological age cannot be estimated morphologically. I hope that determining if methylation patterns in different bone types vary at target positions for age estimation will be useful for future studies and help when applying existing tools to real cases.
