Ep. 8 Identical Twins, drugs, smoking, and crime. Applied Epigenetics with Dr. Athina Vidaki, Erasmus MC, Netherlands

[00:00:00] Angela: Hello and welcome to Secrets from the Crime Lab and educational podcast, where we discuss forensic science and related scientific fields and topics. I'm your host, Angela Swarts. And today we're going to continue on our discussion from last week where we were talking about phenotyping in a forensic context. And today we're going to expand that conversation into the field of epigenetics and epigenomics, which very basically, it's just the study of how your behaviors and the environment can cause changes in your phenotype. And with me today, I have a lovely guest, Dr. Athina Vidaki. She's an assistant professor of Applied epidemo epigenomics at Erasmus Medical Center in Rotterdam, Netherlands. Thank you for joining me Athina.

[00:00:50] Dr. Athina Vidaki: Thank you very much for the invitation. I'm very happy to be here and talk to you.

[00:00:54] Angela: Can you tell us a little bit about what is epigenetics, epigenomics and what it is that you do? 

[00:01:02] Dr. Athina Vidaki: Yes. Sure. So, um, epigenomics or epigenetics are two terms that are used interchangeably basically they mean the same thing. Although epigenomics is usually used when we look at the whole genome and the whole sequence, but epigenetics is a relatively new field, I would say, compared to genetics, especially in forensics, but very exciting. And, we see that, it's expanding quite rapidly, with various applications, methodologies, and so on. epigenetics, I would describe it as, an additional layer of information that our DNA has, which is not a read by the sequence. But it is rather read by the chemical modifications that our DNA has as a response to different environmental factors or, different conditions that our cells have to respond to.

I was, attracted to epigenetics early on during my PhD at King's College in London, already a few years ago. At that time, epigenetics was very new. But I was very interested to, look at, some first applications and, I discovered the huge potential. And then I moved to Rotterdam, for my post-doctoral research. And since one year now I lead my own group and, we are all working together in this and we have a, quite a focus on the technology side of things, because of course, with every new biomarker, we first have to make sure that we have the right tools to study it.

[00:02:28] Angela: It's new, especially new in forensics. I would say it's not really used much yet because there are some complications when it comes to applying it in a forensic context. But, when we're talking about all of these factors that can, add this extra layer of information on our DNA. It has a lot of effects, on our health as well... illnesses, behaviors. So there've been links to epigenetic mechanisms in cognitive dysfunctions, respiratory illnesses, cardiovascular, reproductive, auto-immune. There's a big epigenetic component in cancer and stuff. So in your research isn't necessarily just isolated on forensics, it touches on more than that. Yes? 

[00:03:17] Dr. Athina Vidaki: Yes. Yes. Especially lately. We are also seeing the potential of some of the assets that we are developing also for clinical applications. Yes, that's true. so I think, one way that I usually put it to my students, and maybe it helps also our listeners is that basically epigenetics is, something like our clothes. So the clothes of the DNA. So if it's, cold outside, you wear a jacket. If you're at the beach, you wear a bikini. Um, the DNA itself has the same abilities. It really can package itself, and it can really modify itself to respond to which genes need to be transcripted and translated, what is really needed at every moment. So this is very dynamic. There are of course regions that are protected and are inactive, at all times, but there are a big portion of RPG now that is very dynamic and very changeable over time. Just by looking and thinking of this definition, of course, epigenetics can be so informative. So the first kind of, applications and, advantages of using this epigenomic marker is in disease and in health. Of course, we see that, a lot of effects, especially early on in our life can determine certain epigenetic patterns that you can read throughout your life and it gives a lot of, potential not only in the detection of the disease, but also in prognosis and classification. so it's used really, very widely.

[00:04:41] Angela: And things that you do as an individual can affect the epigenetic factors on your DNA. 

[00:04:50] Dr. Athina Vidaki: Yes. Yes, absolutely, it is almost like a log of your life. And I think the first, or at least the very popular application in forensics, which is the age prediction, says exactly that. So just thinking that we can investigate a few letters, a few CpGs, this is the biomarkers, how we call them. If your DNA methylation biomarkers, and we can quite accurately predict how old you are. It tells you that this mechanism is really very general and it has this ability to really give an indication about how you live your life and maybe what the effects in a very general way you can read on the DNA.

[00:05:33] Angela: And I think that some of the, Critics of forensic applications are concerned about those things about, you know, because you can look at it, look at these factors and there's some privacy concerns around that. You know, if you were a chain smoker or maybe you can, can you tell somebody was doing perhaps some illegal drugs maybe as well? There's privacy concerns just, and medical. Like you can tell some medical factors as well. But what are your thoughts around the privacy issues? 

[00:06:04] Dr. Athina Vidaki: Yes, I think it will be nice if I start with a little bit of a small summary about what we can actually, read first and then I'm very happy to. So, um, a few years ago, we kind of seeing all this potential of epigenomics, and looking how well epigenetics can be used for age prediction we thought, okay, what else can we predict based on this, uh, markers. And, we wrote a nice perspective article together with Professor Kayser, on, what else can we tell. Especially in terms of life, lifestyle factors and environmental influences on our, on ourselves. And for example, smoking, we realized that there are a lot of studies where they, have identified suitable marker sets, basically change according to whether you smoke or not. Or for example, alcohol consumption that can really, have been investigated that much, that we can already use them for prediction. However, of course, as tobacco smoking or alcohol, they are chemical substances themselves. We can also, maybe, move towards also other substances, such as illicit drugs. We are very, at the beginning in this research. There are, it's very difficult, of course, also to do such studies and control illicit drug abuse in individuals and volunteers, but in the very few studies that exist out there we see that there are some effects, but we haven't reached yet the point where we can predict. Of course, because toxicology is also very good, for, for determining this, but we can take it even a step further and start thinking about diet or, body size and shape for like a BMI, like the body mass index or how active somebody is or where even on the planet they live. So we could think of does the water we drink change our epigenome? We know, for example, the sun exposure change the epigenome of our skin. So it starts making you also think, how can we use, even in the very far future, the epigenetics to predict these things. But, most of these factors are still very, very, early. And it's good to have these discussions now, of course, because I agree with you that there are a lot of ethical and privacy issues which need to be discussed in the community and, assess whether, knowing any of these factors is actually useful for forensic applications. And if we decide that the answer is yes, how do we make sure that they're used, in cautious and appropriately.

[00:08:31] Angela: Yes, because, I would think that a lot of these things, yes, when you're talking about a criminal investigation, additional information, especially when you don't have any other leads is always going to be wanted, but there's no way to know necessarily whether or not that information is going to be probative to an investigation until you follow down that road. But then if you go down that road and it turns out that it wasn't related, did you just waste a lot of resources doing all these epigenetic tests ... for nothing? Cause it is a lot of expense. It's not an inexpensive test to do. 

[00:09:10] Dr. Athina Vidaki: No, this is, these are expensive tests and this, our test also though we still don't have, so I can predict that, of course, the value of some of these factors is not the same. I would, for example, think that predicting somebody's age, could be really, really useful and a very good investigative lead, similar to, for example, um ancestry, because it's just so distinctive and it's something that is a factor that can really help narrow down suspect pools. However, there, we also have the effect of general health and lifestyle on your biological age, which is not necessarily the same with chronological age. But I would think that, it would, justify the expenses and the time invested. When we talk about lifestyle, this is a different story. And I think, yeah, I myself don't know if this is a, is it, this is gonna turn out to be useful for a forensic investigation, but sometimes I guess if you have no lead, any lead would be, useful.

[00:10:08] Angela: It's It's something it's information, especially if it's a case that's very cold. You mentioned something about, you've mentioned age a few times and that there is a difference between your chronological age and your biological age. And, I don't know if you're familiar with it, but there's a professor of genetics at Harvard, David Sinclair, who does the Lifespan podcast. He studies aging specifically. And he talks about different types of lifestyle changes that you can make to change your biological age. And he said for himself, by changing some of these lifestyle behaviors that he reduced his biological age by about 10 years. So if that type of thing is possible, how accurate is that going to be for us for forensic purposes? If we're plus or minus 10 years, I mean, how accurate is it? 

[00:11:03] Dr. Athina Vidaki: No, this is a very good question. So I think at the moment, by looking at a few of these markers, because of course in forensics, we always try to focus on the very good informative ones, rather than looking at the entire genome, we can at the moment predict somebodies, chronological age with an error of, I would say, plus minus three to four years, things are getting better and better with time as we discover more age informative markers. So I have seen models that go even lower, down to maybe plus minus two years. But of course these models are very tissue specific. So a model that will work in whole blood might not work in saliva or in sperm or in other body fluids. This is very important to have in mind. Also, at the moment we don't really know what we are measuring with those models. We know that our DNA methylation is very nicely associated with age, but we are still, to discover mechanistical insights and really understand the function behind it. And why our DNA methylation is age associated. Now of course there are those studies that's yeah, it's a very natural thought thinking if I test my own DNA and actually I haven't done it yet, and I discovered that I'm five years older. Can I do something to bring this down lower? And of course, if one, eats healthily and is active, like we know from our, from our doctors, you could reduce the chronological age. But I would be very cautious with making harsh conclusions on this because we are still into the development of the technologies. We still need to understand the biology behind. And we also don't have many longitudinal studies yet where we follow and we track people on, on their life and how changes in lifestyle change the age. I do believe that this is true, but it's difficult to comment on how generalize this is, how specific and how stable as well.

[00:13:04] Angela: Right. 

I can give you an example maybe on the smoking We know, because it's just a, such a distinct trait. We have some evidence so far that if you are a smoker and you quit and we reanalyze your DNA maybe in five years time, 80% of the smoking signature that you have will be reverted back, but 20% will still probably be there to remind you of your mistake forever. And of course it also allow us to predict, former smokers, and so on. So life is there to protect us and our cells and our DNA's there to protect them, so of course, once we start changing our life, we will be able to see that on the DNA to an extent that our technologies allow.

[00:13:51] Angela: The smoking is interesting because one of the one of the types of evidence that I've seen quite frequently in casework are cigarette butts left behind at scenes. And the number of times a cigarette butt is that investigative lead, I probably can't even count the number of times I've searched a DNA profile from a cigarette butt in a national database. So it's a very good piece of evidence. And if we don't have any leads and we didn't get any database hits, looking at the phenotype, and the ancestry, and these epigenetic factors could definitely be useful. But one of the things that the smoking made me wonder about, so you can tell if someone is a current smoker, you can tell if someone is a former smoker, what if someone's not a smoker, but they've lived with someone who is a smoker. 

[00:14:42] Dr. Athina Vidaki: Yes, this is something that we've wondered ourselves as well. Um, so, um, at the moment, we can predict smoking habits with quite high accuracy. But I would like to already mention that this is very heavily dependent on the amount of tobacco uh, smoking, in the intensity of the smoking itself. And also not only how many cigarettes, a person smokes a day, but also for how long. So you can already see that there are some dose-depending effects and that we see it in blood. Now, passive smoking is very interesting, because in a way, passive smokers do smoke some of the already inhaled smoke from a current smokers. But there we are still in the dark. We don't know exactly the effects yet and how this will affect the predictions, but this definitely one of the things that we want to look into. In babies, where, for example, the pregnant woman smokes and the baby's still in the belly we know that when they are born, we can see some of these smoking signatures also in their adult life. But of course, that's not exactly the same because of course the baby's in, in the same, body but we expect that there will be some effects also in the passive smokers, we just haven't investigated them fully.

 So, if you were to give information on smoking habits to investigators, you would have to definitely qualify that information pretty heavily, right? Because you wouldn't, you might be able to tell the difference between somebody who is a heavy smoker, but someone that's quit smoking or been a passive smoker it might be more difficult to differentiate this 

[00:16:30] Dr. Athina Vidaki: Absolutely. And this is actually where we are at the moment. So we have a model that can predict with quite high accuracies and we are testing different scenarios now in different people that have quit at different times, have they started smoking also at different times, and see how this prediction, predictions change. But indeed, such prediction can not come with a yes and no answer. It should come with probabilities on the smoking habit, which will take into account all these factors. So we call the so-called pack years, which means how many cigarettes you smoke for how, how long. And then we can even be even more complicated and think about, pipes or other types of, or electronic cigarettes and how this would really affect also the epigenome. These are studies that are still ongoing, and are very relevant of course, because there are just so many smokers worldwide. I was reading yesterday, actually, in 2019, they were 1.1 billion smokers worldwide. So that's scary.

[00:17:34] Angela: I'd be curious if you can cross check those against, changes for lung cancer. 

[00:17:40] Dr. Athina Vidaki: Oh , we know smoking is one of the leading cause of disease, directly or indirectly. And, it's of course, very, interesting to also study the effect of smoking also on other factors like age. So do smokers age faster than non-smokers? And these are all things that we are planning to, to work on.

[00:18:02] Angela: That's interesting. Cause it's just from your life experiences, you have some anecdotal information, but anecdotes aren't evidence. 

[00:18:09] Dr. Athina Vidaki: Yeah.

So we mentioned methylation. We should probably tell people what we're talking about when we talk about methylation. 

[00:18:17] Dr. Athina Vidaki: Yes. That's a good point. I sometimes assume that. As we said, epigenetics is the field that studies modifiable changes on the DNA that are not depending on the sequence itself. DNA methylation is just one of the mechanism of epigenetics. We can also have histone modifications or other type of chemical tags, but DNA methylation is the one that they studied directly on the DNA. Where do we find methylation? The vast majority of these methyl groups, are, added to cytosines that are followed by guanines. And this is where I mentioned before the CPG sites. So cytosines that are followed by guanines specifically have this potential to be methylated or not. Of course, this is the rule and this is what we know so far, but, we also know from very recent research that we can also have some very low levels of methylation on non -CPG cytosines. And we could also see other types of, methylation, derivatives, such as hydroxymethylation, which is for example, very, found very much in brain cells. Yeah, who knows? Maybe we have methylation also in other bases in lower percentages. Like we see, for example, bacteria and other organs have, but we don't know them yet because we don't have the right tools. So CPG methylation is by far the most studied DNA methylation markers.

And just to remind the listeners that aren't as familiar with DNA, there's four bases in DNA, adenine, thymine, cytosine and guanine. And these are the four letters that form all of the codes that give us the instructions from our DNA or factors that control the expression of what our genes code for. So with that little lesson behind us, I wanted to jump back to some of the things we were talking about, lifestyle and smoking, and we're mostly talking about markers associated with tobacco smoking, but tobacco is not the only thing that people smoke. And of course there's been, depending on where people live, the legislation with regards to cannabis smoking is very different and there's usually some type of allowance for the use of cannabis for medical issues. So do you know anything about how cannabis affects the epigenome?

There are more and more studies on this. We haven't studied this ourselves, but they do show that, of course they're also CPG sites or DNA methylation markers that are specific to smoking cannabis. And you would assume that actually some of them might be also shared with the smoking, tobacco smoking, markers. Certainly whatever we use either it is, we smoke it or we inject it or, prescription drugs or illicit drugs. Any substance that we take will leave their mark on our DNA, that's for sure. So I can even generalize this, that I think every drug or substance that we take will leave some sort of, yeah traces behind. 

[00:21:19] Angela: Yes, but we got to figure out how to find those traces. Right? That's the complicated thing right now because the technology is still relatively new. 

 For drugs, I'm extremely interested to, to work on this topic, also because we know people that have studied this so far are more coming from the medical field, trying to understand addiction and how the different drugs affect your DNA, your expression, and all this mechanisms, which are fascinating. But I think for forensics, we are a little bit slow, also because we have toxicology, It's very sensitive. it's very, useful for evidence that we know, are collected from the sample immediately from, from a person. But when we talk about epigenetic profiling of drug use, we talk a little bit more about the habit, more about the long-term, exposure. And this will be interesting also when, yeah, we want to cross check claims. For example, in the medical settings, when they need to do liver transplantation, it's interesting for them to know. This person says they've stopped drinking or heavy drinking. Is this true? Can we verify it because of course a patient that wants this might lie because they want to save their lives. And this could also be, for, I don't know, maybe for forensics, for maybe for crime prevention, maybe for offenders that do take drugs and they follow this programs, rehabilitation programs. So the forensic application there could... Yeah, we have to think about where could this be useful?

[00:22:51] Angela: I can see how it would be useful for people who are going through rehabilitation programs and especially if it's a court ordered rehabilitation program and the court's trying to determine whether or not this person is being truthful. Drug use sometimes, it is time dependent on whether or not toxicology can detect it in the body fluids. So that is something to keep in mind. So if someone says, no, I haven't been doing it. And they stop just long enough to prevent it from coming up positive on the toxicology test. But if you can tell current use versus former use and the epigenetic profile that could be useful in that sense. The other thing I was thinking about is a lot of times we'll find bongs or crack pipes at crime scenes. And those are the items of evidence, much like cigarette butts that would, we would be using to try to get an investigative lead. I can mostly see applications for this technology in no suspect cases where we don't know who the perpetrator is and we're just trying to find out some information. But then when you know, it's one thing, if you get your evidence from a cigarette butt, that's left at a crime scene. It's another thing when you're getting it off of the crack pipe. And then you back that up with the epigenetic profile that shows that someone's been using crack cocaine, for example, and then you have concerns about whether or not that those lifestyle issues are going to be prejudicial once a case gets to trial. So it was a lot of those factors, those things like... We can figure out the technology to find out this information. And what effect is that going to have downstream?

[00:24:34] Dr. Athina Vidaki: That's a very valid point. I think these are all things that it's nice to start discussing and discuss also with the, with other scientists from different disciplines and also from the forensic casework, scientists themselves and the police. We are extremely interested in understanding the effects, the interrelationship between all these factors. For example, we know that the age prediction changes heavily with substance abuse, actually changes heavily even with heavy physical activity. There was one study by a forensic group recently where they showed that elite athletes actually are predicted much older than what they are because of all the efforts and the stress they put in their body. So for us, just the fact that we can investigate the effects that we have on the epigenome, we understand and can interpret our epigenomic data much better. But if it's useful for prediction in certain applications, this is still to be determined. And I think we need to open the discussion on this.

[00:25:36] Angela: Yeah, So it's interesting. So if you're doing an epigenetic profile to try to generate these investigative leads, it sounds like you can't, you wouldn't just look at one factor. You would have to look at multiple factors and then each one of those factors has to be qualified with limitations that are specific to it. So age prediction, like you just said, is affected by stress levels from heavy exercise, and then you've got your lifestyle choices as far as smoking, drinking, et cetera. So it's definitely interesting. And of course, geographical, you mentioned geographical predictions earlier as well. So I know that I live in a very sunny climate, so there's been an increase in the number of freckles on my face.

[00:26:25] Dr. Athina Vidaki: Yeah, that's definitely an effect. I am an expat myself and, I often wonder, would my epigenome be the same if I was living in sunny Greece, has it changed that I live in rainy Netherlands? Can you tell that maybe I'm not only Greek, but that I'm a Greek living abroad? I've often thought about this, this questions we are so early in this. There are some studies that show, ancestry specific DNA methylation, but we do not know why or the mechanism behind, but it's definitely something that would be very general. It would be very hard to study and point like account for all these other factors that can change your epigenome between your participants and how do you control for them. And maybe I can comment also on your first, comments with kind of this big epigenetic fingerprint, because I have like this in my head, we have an epigenetic fingerprint. It can give us information that all these factors, this is really what we dream may be to have, in a few years, or maybe more than a decade at the moment that technology really allow us to look at just a few markers every time. So a lot of our assays are small scale, 10, 20 markers. All our models are basically very small scale. So if we want to predict all of these, we need separate tests. And this is why we put a lot of effort on the technology development that will allow us in the future to study hundreds of markers from forensic traces, because this is not isn't available at the moment.

[00:27:54] Angela: It's limited in what we can accurately predict now. It's expensive. And so we need to be very careful on when we decide that it's going to be worth the time and investment. And is it going to give us information that's going to be helpful in a forensic context? So with all that in mind, has this technology been employed in forensic investigations that you're aware of successfully or unsuccessfully, both are important. 

[00:28:28] Dr. Athina Vidaki: I think that, forensic epigenetics is still very at its infancy. I know from collaborators, around the world that they have tried to use age prediction tests, because this is really the application that is very booming. A lot of people are trying this when we have large amounts of DNA and cases allow to perform age prediction, but I'm not aware of any case epigenetic data has reached the court. Because we of course are talking a lot at the moment on DNA investigative intelligence and DNA phenotyping. Of course, this is simply leads to the police and the police based on the science and the advice of the people that are experts should judge if the investment is worth it. But epigenetics can also give evidence that is actually useful in pointing towards the person, or the tissue type that is useful for body fluid identification or maybe for discriminating identical twins, which is by far my favorite application, but by far the most complex one as well.

I guess it's interesting identical twins are something like you've always been taught like identical twins, their DNA is identical, Well, not really. 

[00:29:43] Dr. Athina Vidaki: Not really.

[00:29:44] Angela: It depends on what test you're using. Yes, our normal forensic DNA profiling that we do looking at STRs, identical twins, it will be identical. But when we start getting into the actual sequence, it's different. We have the single nucleotide polymorphisms, and now we also have epigenetics to look at as well. So no twins aren't exactly the same. 

[00:30:09] Dr. Athina Vidaki: Yes, identically different I would call them. But the problem here is that indeed STR profiles are basically identical. I think there was one report that I could find online with one special case where the twins differed in one STR that must have been extreme luck or, yeah, something very rare. But, also when we think about specific nucleotide changes, letter changes in our genome. These are very few. So if we think of our genome is 3 billion bases or 3 billion letters, they could find on average around four to five such changes. And of course these changes will be different between different twin pairs. So one had to really look at the entire sequence to be able to identify which ones can I analyze in my trace sample. And this is very costly. I am aware that there are some cases that people follow this approach but it's very hard to have this data in court because the methods haven't been validated. It's difficult to be able to convince the court that we are very sure about these technologies.

[00:31:13] Angela: I have to say that there've only been a few cases that I'm aware of from my experience where identical twins were an issue. And in one of the investigations, it turned out both of the twins were involved in commission of the crime. It wasn't really an issue there. And in the other one, one of them was incarcerated at the time and the other one was free. So there are other ways to do it, but it'd be really cool if we could advance the technology so that, we could not have to sequence the entire genome to find these few little differences. 

[00:31:49] Dr. Athina Vidaki: Absolutely. And I hope that epigenetics will soon give us something or at least we have to think out of the box here. I did a little bit of, cause I was also wondering myself, how rare are these cases? And, they are rare, of course we know that one, every 250 births now results in a identical twins.

So they're not so rare, but you will be surprised. In the Netherlands, in 2017, I did a 15 year look at the database with the help of the Netherlands Forensic Institute, and we actually could find 300 unique cases, from burglaries, sexual assaults, and, yeah, even one murder. And there you could even see what types of traces we have. We used really this information to guide our research. We have published a bit on this topic and, I don't know for me, justice should be universal. So I know these cases are rare, but we should be able to, give a solution and be able to point, towards the innocent person, at least, because in those cases, yeah, perpetrators walk free because we can't, we cannot, yeah.

 Well, I mean, the question about identical twins is one that I hear all the time, so it's definitely worthwhile to have the tools available to us to be able to determine, actually the true identity or the true source of the biological material. 

[00:33:13] Dr. Athina Vidaki: It is very difficult, unfortunately. So some of our first studies, when we look at a big portion of the epigenome in twin pairs, we could find some differences in the DNA methylation markers. These were very also twin pair specific and sometimes they were also driven by disease or by different environmental effects. I often also wonder it would be nice if once twin smokes and the other doesn't, and then we can use maybe smoking predication for this, 

[00:33:41] Angela: Or maybe one twin lives on one side of the world and the other twin lives on another, and then you can see, are there location differences? 

[00:33:48] Dr. Athina Vidaki: Depends the location of the crime, for this. But we did recently conduct a much larger study. We looked at thousands of those twins with the help of our collaborators. And we could point towards some DNA methylation that is, mainly, determined by stochastic mechanism. Or this is at least our kind of hypothesis, similar to what happens with STR perhaps, but also very different. But those stochastically methylated sites, where may be, or are a good source of, using them for such a test, because they will be able to differentiate everybody, including twins. The problem there is that we need many. There are hundreds of them. And each marker has such small effects that in order to build a prediction model or be able to create a statistical framework with likelihood ratios for court, this, needs a technology again that we don't have. So a lot of these epigenetic applications point towards the need for robust standardized technologies which we don't have at the moment.

[00:34:56] Angela: Well, it's, it's interesting 'cause I know a lot of the technologies that we have that are routinely available to us now in forensics were at this point in the past as well. We've got all of this information and that's expensive to get. And then over time we've focused and narrow it down to the absolute essentials because you can look at just for forensic DNA phenotyping for eye color prediction there were a lot, a lot of biomarkers that were examined until they've pretty much narrowed it down to about six. So do you think that we're going to be able to get there with epigenetics as well? Maybe not six.

Well it depends on the application, I would say. So for the application we haven't mentioned so far, which is a tissue identification. I think we could possibly imagine a test only on a few markers. For example, we know that semen versus blood or saliva has a very different DNA methylation profile. So we find those cytosines that are completely methylated in semen and completely non methylated in all other somatic tissues. So I think for tissue identification, we can probably achieve that. We have some tests already there. I know that there are some countries that use this for casework, probably alongside mRNA test or other biological tests. But for the more complex predictions, I would really doubt it. And the reason for it is that for a SNP, we have categories. You can have one letter or the other. But for methylation we work with percentages, is the ratio. How, how many of your cells in a given stain are methylated? So we can have 5% methylation, 80% methylation and so on. And we see that the effects of lifestyle for example, are so small and are so spread across our genome, that probably a few, it would be quite difficult to, to do Although I can say with the smoking, we are at the moment with 13. So with 13 CpGs, we can quite accurately predict the smoking habits, but for more complex traits, that would not probably be possible.

[00:37:13] Angela: Now the body fluid identification is one of those aspects that I've always found very useful now. Because most of the tests, the majority of. Forensic laboratories when they're testing for body fluids, we're using some very old chemical, or immunological tests. And some of them are specific. Most of them are not, they have varying degrees of sensitivity and there are a lot of things that you have to consider when you're interpreting that result. Um, and it doesn't really... when you have mixed body fluids, it gets more complicated. And the value of using our mRNA or looking at the tissue methylations to me, seems like an exceedingly useful thing. So even if that technology is not as robust as perhaps we would like it to be as compared to some of the other tests that we have that have been around longer. I still think that it would be, it's probably better than the serological methods that we're using. 

[00:38:17] Dr. Athina Vidaki: I completely agree. I think that those biochemical tests have been there for quite some time. They have been extremely useful for crime scene investigations, especially when you want to do a quick test on site to know, is this blood, is this ketchup you know, is this useful. But when we talk about very, small stains, very sensitive tools also for cold cases where all we have is maybe some DNA samples is it can be very beneficial to have some molecular methods. And mRNA, for example, has been really useful here in the Netherlands at the Netherlands Forensic Institute, they use it routinely almost, hundreds of cases. I know they, it has been very useful. For DNA methylation, we are still a little bit behind, It would be much harder to look at mixtures because of the percentage nature of DNA methylation. It's not a presence of a marker or not like we have in mRNA, but in DNA methylation, you wouldn't be able to separate the different strands you would analyze. But we are also working on that and we plan to really, again, think of small tricks in the lab where we can use to be able to do that. But for single source samples, it can definitely be useful already.

[00:39:37] Angela: Yeah, a lot of these, I'll call them more investigative molecular tools, they are very... They're better for single source samples, it's better for a neat bloodstain or a neat semen stain. As soon as you start having mixed body fluids for more than one individual it becomes very complicated and difficult to tease that information apart. But it's still good information to know that even though it looks like a bloodstain, it's actually a mixed stain of blood and semen, for example, right? 

[00:40:11] Dr. Athina Vidaki: Absolutely. Yeah, I think it's important to, to acknowledge the significance of the evidence collection itself and how we should like, alongside with thinking about what new biomarkers to use and what new applications to investigate to also never forget that we could and should try to improve. Also the sample preparation, how much DNA we get out and how maybe we can, help us with the mixed samples and the small traces. I think this is something that, should go alongside with all the marker discovery part

[00:40:45] Angela: So what other complications do you have based on... There's couple of things that come to my mind is like, there's no such thing as a pristine sample from a crime scene, right? They're all compromised in some way. And the amount of biological material is often extremely limited. And how does that affect the ability of this technology to get useful information? 

[00:41:12] Dr. Athina Vidaki: Yes, Um, well, the, the number of challenges that we have in epigenetic profiling are many. So the first is the heterogeneity of the sample itself. So if you think that, if you think of genetics, we know that we have the same sequence in all our cells, but epigenetics is in theory, different in every cell. So for example, I analyze my blood sample today and my blood sample tomorrow. If the proportion of the white blood cells is different, my methylation profile can be different as well. So the cell composition and how we deal with it is key. And also we should always have matching samples when we compare a reference with a crime stain. It's very difficult to have a sexual assault case with semen samples and try to match it with methylation from a buccal swab. This would not be always possible. So that's a very big technical limitation. I can also talk a lot about the statistical modeling and how we discover our markers and how we interpret that but I won't go to there. I will tell you that the methylation detection itself it's also, quite problematic at the moment. So most of the epigenetic assays that we have published are based on this first step of chemical conversion. So we have to somehow translate our methylation signals into sequence variation that we can then go into the PCR and amplify it. And that process is extremely, um, damaging. Uh, it's it's so-called bisulfide conversion. And this one is, requiring a lot of sample. We are talking certainly, at the nanogram level, even hundreds of nanograms. 

[00:43:03] Angela: Oh, my gosh. 

[00:43:04] Dr. Athina Vidaki: Which is never available of course, in forensics or most of the time and therefore sensitivity is also the second biggest problem for me. So we need to find ways to change our, methodology, to be able to handle such, such amounts. And of course, even if sensitivity goes down, if this, the chemical process we use damages the DNA and fragments it even more, that's also quite a problem. So we are, we are in the process of trying to, work with this, with this issues. Finally, from the technical side of things, I would also mention the differences between the methylated strands. Because again, we look at a population of cells, we extract DNA and we amplify it. If some strands are methylated and some are not, they compete with each other. They have a different sequence after this conversion method. Therefore, when you amplify it, you can have amplification bias. So how accurate is really the DNA methylation we, we detect. And that's something that I believe researchers quite underestimate at the moment. We need to validate our results and we need to really be more cautious on the methylation signals that we use for our models and for our interpretation.

[00:44:20] Angela: It definitely sounds like a very complicated minefield of a lot. You've got your work cut out for you. 

[00:44:31] Dr. Athina Vidaki: I am very surprised because, like every time I talk about epigenetics, actually, this is what people conclude. Oh my God, this is such a complicated field. Why are you, why are you so passionate about it? But I think every complex problem can be transformed into simpler problems. As long as we have the right data, it will take more time. It's not a field that we can apply tomorrow, but I think once we reach there and we really understand how to interpret DNA, methylation data, we can get a lot of interesting information out. It might take a few years, but we will reach there.

 I think if we put it into context, when we first started just doing DNA identification, it seemed like this is super complicated and we're never going to get there. And, oh my gosh, there's so much information. How do we know what we're going to look at? And now we've got it very fine-tuned. And to tell everybody how quickly that happened. It is an ongoing struggle for people in the field to keep up with the changes in technology, because they are happening so quickly. And I often have a lot of a lot of sympathy for people who interact with, these applied genetics, applied molecular biology fields, because, toxicology is pretty stable. Drug testing is pretty stable. When you get into things with DNA and RNA is oh no, that, that was last year. It's different now, so people who thought they had a grasp on it, I'm like, oh no, it's changed again. 

[00:46:08] Dr. Athina Vidaki: Absolutely. And this really highlights also the need for forensic biologists to keep up with the field and for finding ways to get quick accreditation's and key, quick validations, and really like it takes time. And we have to also think, how can we keep up with this development, which is wonderful because it means that maybe in a few years' time, we can have, we could have solved a lot of the issues that we encounter today. I'm sure people thought that, RFLP profiles, were very complicated, but they were very successful. So... 

[00:46:46] Angela: Yes. 

[00:46:46] Dr. Athina Vidaki: Epigenetic data will, and generally DNA data I think will, come to that stage at some point .

[00:46:52] Angela: Yeah, I have to think back to when I started in this field, there was no such thing as an automated DNA sequencer or genetic analyzer, and now that's everything everyone has. You know, I remember. manually pouring gels and using radioactive material to label my DNA. And a few times where I had to go into a minus 20 freezer to load my gel, just cause we needed to slow down the migration for our particular tests. So we don't have to do that anymore. We get to actually work in, properly air conditioned or heated laboratory spaces and we're not working with radioactive things most of the time anymore, or at least not in forensics. 

[00:47:37] Dr. Athina Vidaki: Luckily. absolutely. And I think, in order to keep up with these developments, we have to really not do it alone. And I think conversations about, for example, lifestyle prediction really do showcase the importance to talk with social scientists, with ethicists, with psychologists, with lawyers, with policy people, and, we have to make decisions together about. Should we invest in this or not? What does society think? And it's not all about the technologies, but also how we use them. So it's very important to change our mindset of more interdisciplinary research when it comes to future DNA.

[00:48:17] Angela: Absolutely. I definitely agree with you on that, which is why last time I talked to Dr. Matthias Wienroth and Dr. Carol McCarthy about those topics. And so. talking about the epigenetics just definitely seemed like the next step in that as well. And I know that you do a lot of collaborations and that's essential, and of course you have quite a number of publications as well. And we can link those in the show notes for anybody who's interested in reading up more about this technology and the research and work that your laboratory has been doing. Are you going to be speaking at any conferences or anything in the near future? 

Yes, actually, I'm very excited. We, hopefully should be able to travel again and meet each other in person. So I'm scheduled to give a presentation, on forensic epigenetics in general, like a nice overview of the field in the GRC conference, in June, And, also at the ISABs conference of the International Society of Applied Sciences in Dubrovnik, in Croatia also in June. And, we have just submitted our abstracts for the ISFG for the International Society of Forensic Genetics in Washington, in September. So yeah, I hope we will be able to present our work there.

I hope that one's accepted. Cause that's the one I'm hoping to get to go to fingers crossed depends on scheduling. and stuff, Thank you very much for taking the time to come on and talk about this exciting field. I wish we had more time, cause I think that you know, I personally would love to understand more about the statistical models behind all of these things and choosing things, but that might be a little bit deep and difficult to do on an audio podcast as well. Did you have any closing remarks you wanted to share? 

I think that, it is important to, to understand that this, epigenetic field is slightly different than the genetic, because we don't only work with DNA sequences, but we also work on chemical tags and profiles. So yes, it is complicated. But, it shouldn't yeah. disappoint or stop everybody, from looking at it as, one of the future fields and one of the lines in forensic DNA testing that one day can be very promising. And of course I would like to thank you for giving me the opportunity to be here today and chat with you and your listeners. And yeah, I was, I enjoyed our chat really very much.

[00:50:45] Angela: Oh, my pleasure. You're welcome back. Anytime you want to talk about something nerdy. 

[00:50:50] Dr. Athina Vidaki: Super. I will take that into account. Alright, thank you so much.