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What if we could actually identify and even correct significant autism risk factors in utero, before a baby is born?
This is the promise of the game-changing research of Dr. Janine LaSalle, epigeneticist extraordinaire from UC Davis. With autism rates continuing to increase at alarming rates (the latest data from the CDC, released in 2023, is 1 in 36 children in the US, an increase from the prior 1 in 44), new ways to prevent and treat this condition are of utmost importance. Dr. LaSalle is on a mission to change that, by investigating something you probably haven’t heard much about yet – the placental methylome. Methylation signatures in the maternal placenta are yielding information both for predicting autism in children and for intervention targets to prevent the development of the condition. It’s just incredibly exciting.
In our conversation, you’ll also learn how placental methylation behaves differently to DNA methylation in other tissues, and how the placental methylome may also help us better understand cancer as well as mechanisms that restore more youthful epigenetic patterns. Hold onto your hats folks – yes this is deep science, but it’s also actionable even now. You’ll hear how methylation support specifically in the early pregnancy window is key and how it’s also protective against insults from environmental toxicants. You’re in for a treat with this fascinating conversation that has far-reaching implications for you and your patients. ~DrKF
Towards Predicting Autism in Utero with Dr. Janine LaSalle
The placental methylome is the landscape of methylation marks on DNA cytosine residues that exist in placental cells. New research, conducted by Dr. Janine LaSalle and the team at her lab, suggests that the placental methylome can provide unique insights into the fetal origins of disease, including autism, and offer new ways to change the trajectories of their manifestation. Part of these insights relate to the way methylation behaves differently in placental cells compared to other cells.
Dr. LaSalle and her team have identified a new gene – NHIP – which when altered is strongly associated with autism risk. This exciting discovery paves the way for potential future therapies to bypass this alteration and reduce the high rates of autism in children. Not only that, her science as well as that of her colleagues, indicates protective effects of prenatal multivitamin use in very early pregnancy (i.e. the first month, a time when most women are not yet taking a prenatal multivitamin), both against the effects of environmental toxins and against autism itself. This progress is sorely needed to help alleviate the burdens and risks of the disease, as well as the associated psychological, economic, and societal costs.
In this episode of New Frontiers, learn about:
- The placental methylome as a potential surrogate marker for brain development and implications for predicting and preventing neurodevelopmental disorders.
- How methylation in the placenta behaves differently to methylation in other tissues, acting more like a “signature of expression” than regulator of expression.
- The discovery of a genetic alteration on the newly-identified NHIP gene that when homozygous is associated with autism risk.
- How this paves the way for potential future therapies such as peptides that can bypass that genetic defect.
- The potential role of prenatal vitamins in protecting against autism risk, noting that the window for protection is both incredibly important and also small and early.
- Low rates of early maternal prenatal vitamin use suggest there is room for much improvement and impact.
- The potential U-curve of risk with too little/too much folate or B12 in early pregnancy (although sufficiency is always important).
- Maternal obesity as a risk factor for Autism Spectrum Disorder.
- The influence of toxin exposures, particularly PCBs, PFAS, and air particulate matter.
- Implications of the identified gene for clinical decision making, particularly in supporting antioxidant status and addressing toxic burdens.
- How methylation support can be potentially protective against environmental toxicants.
- Shared characteristics of plasticity between placental and cancer cells that may provide new insights into addressing cancer, or even harnessing safe cellular rejuvenation?
- Characteristics of polycomb regions of the genome and how they don’t use methylation in the same way as other regions.
Dr. Kara Fitzgerald: Hi, everybody. Welcome to New Frontiers in Functional Medicine where we are interviewing the best minds in functional medicine. And of course, today is no exception. I am beyond honored and thrilled to be talking to Janine LaSalle. Let me give you her background, and we’re going to jump right into what will no doubt be one of the best conversations we’ve had on New Frontiers in some time. Dr. LaSalle is professor of microbiology and immunology at UC Davis, with expertise in epigenetic mechanisms at the interface of genes and environment. Dr. LaSalle serves as the deputy director of the UC Davis EHSCC, which is the Environmental Health Science Center and co-director of the Perinatal Origins of Disparities Center. Her lab uses genomic and epigenomic technologies to investigate DNA methylation signatures in autism spectrum disorders and beyond. Her lab utilizes perinatal tissues such as placenta, cord blood, newborn blood spots, and cell free fetal DNA for epigenetic prediction and prevention of health disparities. Dr. LaSalle, welcome to New Frontiers.
Dr. Janine LaSalle: Thank you. A pleasure to be here.
Dr. Kara Fitzgerald: Everybody who’s just heard your bio is incredibly excited already. I can tell you’re just doing some extraordinary work. We were talking about how I heard you on the Active Motif podcast. If anybody wants to listen to a really dense, scientific discussion of Dr. Lasalle’s work, hop over there. It’s October 2023, but I heard it at that time and was just really hoping that you would consider joining me. In 2017, I actually interviewed Dr. Moshe Szyf, who went on to become a mentor of mine. And in 2017, he said, very extraordinarily to me, that he anticipated us being able to actually diagnose autism in utero. And it was such a remarkable statement to hear in 2017 and something of such promise. I was deeply moved by that and his suspicion that the interventions might allow us to actually correct it in utero before it even presents.
Dr. Kara Fitzgerald: And that was momentous. It was quite a statement and very moving, and the hope that came with that was huge. So, when I heard your work, that you in fact were bringing this to birth, to use a good metaphor. His promise back then just wowed me. Of course, we’re going to drill into this, but I want to know how you got into epigenetics in the first place and then we’ll move into talking about how you mapped the placental methylome.
Dr. Janine LaSalle: Yeah. So it goes back about three decades, I think. I was a graduate student in immunology, and at the time, I knew I wanted to do a postdoc in the Boston area but wanted to maybe explore just outside immunology. I met Marc Lelande at the time, who was at Children’s Hospital in the Howard Hughes Institute. It was interesting because we were collaborating with him, and I was cloning T cells and he wanted to collaborate, and I thought he was going to send a postdoc or a grad student, but he shows up in the lab next to me in the tissue culture and I started learning how to clone T cells. In the process of getting to know him, he said, “Oh, I’m looking for a postdoc, would you consider it?” Then he started telling me about this crazy Prader-Willi and Angelman syndrome. I never really liked genetics that much as an immunology graduate student because it was very dry, but I just got fascinated by, okay, how does this occur, and it was really interesting. So, yeah, he converted me into an epigeneticist around ’93, I guess. At those times, when you would go to genetics meetings, epigenetics was the quirky exception to the rules. It was the people working on imprinting, X inactivation, and maybe thrown in with the mitochondrial people, right? The quirky exceptions to Mendelian inheritance.
Dr. Kara Fitzgerald: The methyl group on the cytosine was sort of something to be ignored. Is that-
Dr. Janine LaSalle: Well, they knew about DNA methylation then, but it was not really… Yeah. I mean, we knew that was one of the marks of imprinted regions. That was known about even when I started. I was doing more of the microscopy and Fluorescence in Situ Hybridization to look at replication timing at that time. But, yeah, methylation was in its infancy. We didn’t really have a human genome yet. So it’s been just really fun for me over the decades to see the field expand and so many people get excited about epigenetics and how it’s not just a quirky exception in a few rare human genetic disorders. It’s actually a rule of thumb for most of our complex disorders.
Dr. Kara Fitzgerald: What a full flip, right? Would you say that that was borne out of… I mean, we often talk about it in functional medicine that this movement towards really lighting on epigenetics was kind of born out of mapping the human genome and then seeing that, whoa, it’s not the Rosetta Stone we thought.
Dr. Janine LaSalle: Yeah. I think that, again, I remember hearing these arguments about the human genome and thinking, yeah, they’re kind of overselling it. I think it’s exciting because now people are coming around to the fact that genetics on its own only gets you so far and we really need to understand that interface, that connection with how our genes respond to the environment. I like to say the genome is not in a lockbox away from all the other influences, but a lot of times genetics, in its silo, treats it that way, right? I mean, people will just study the genes alone. And again, you know, that gets you at causality, but we realize that causality is not 100% for most genes. I mean, definitely, we work on syndromic disorders, which are, you know, close to 100% penetrant, but not completely. And even those have interactions with the environment.
Dr. Janine LaSalle: We’ve gotten into looking at Down syndrome, which again is the classic genetic disorder. We know it’s caused by an extra chromosome 21. But yet, some kids with Down syndrome have congenital heart defects, and some don’t. So we’ve recently looked at an epigenetic signature that can distinguish those. And we’re working on Rett syndrome. Rett syndrome, because it’s on the X chromosome, you can have identical twins, one that has what looks like autism, and the other who is functionally quite well off and going to classes. So you can get this whole spectrum within the same genetic-
Dr. Kara Fitzgerald: Something clearly genetic is not influenced solely by the change in gene. And in fact, the change in gene isn’t necessarily a predictor of having outcomes.
Dr. Janine LaSalle: Well, it can still be a predictor, but it’s not necessarily always. I mean, yes, a child with an extra chromosome 21 is always going to have Down syndrome, but how severe that Down syndrome is can be influenced by epigenetics.
Dr. Kara Fitzgerald: Are you familiar with the… I feel like Bruce James was an author on it, but I don’t have it in front of me, so I may be incorrect. It was giving a multivitamin to Down syndrome individuals demonstrated…
Dr. Janine LaSalle: Yeah. So prenatal vitamins, that’s something we’ve worked on in several different projects and it’s interesting, because prenatal vitamins are mostly folic acid, which is a major methyl donor in the diet. It’s there for the prevention of neural tube defects, and it’s been very successful for that. It’s one of the most successful public health implementations that’s been done. It is also preventive for Down syndrome but you need to take it in that first month of pregnancy. I’ve been working with Rebecca Schmidt, who’s an epidemiologist here, and she showed that taking prenatal vitamins, again in that very first month of pregnancy, is preventative for autism as well.
Dr. Janine LaSalle: So we’ve now started to look at that as one of the variables when we have a region that is an epigenetic candidate for autism, we’ll ask, okay, is that changed with the use of the prenatal vitamin in the first month of pregnancy or not? And we’ve seen a couple loci where yes, it’s very clear cut that there’s an association, and it’s always protective.
Dr. Kara Fitzgerald: That’s incredibly interesting. And so listen, there’s a lot… We’re skipping ahead to, like, question six and seven here on my list, but I want to ask you and I want to make sure. We want to talk about that gene you just identified because we’re talking… Well, we need to get back to the placental methylome, but these genes you’re identifying are probably some of the ones that we’re going to analyze in utero to determine… And then you’ve just suggested that you might be able to turn off this genetic involvement by the use of folic acid, or in the functional medicine space, we’re more often prescribing actual activated folate versus folic acid, the oxidized version.
Dr. Kara Fitzgerald: But it’s the same point. We’re supporting methylation through a folate vitamer. I just wanted to throw it out there, and then we’re going to get back to all these other questions. There was an interesting Johns Hopkins paper that came out, I want to say maybe 2016, looking at a U curve of serum levels of folate in women who gave birth to kids on the spectrum. So deficiency and excess were both associated. And I just want to get your thoughts on that.
Dr. Janine LaSalle: Yeah, that’s a really good point. Some of that may be the ones that are taking the really high excess could be doing it for medical reasons, so maybe it’s that original medical reason which is why. But I’m also open… We are actually now collaborating with a group here who is looking at the excess folic acid as well, in both mouse models and human organoids. It’ll be interesting to see what comes out of that work.
Dr. Kara Fitzgerald: We’re going to stay on this. I’m telling my team we’ll stick to you like glue.
Dr. Janine LaSalle: Yeah. Because it could be both. The thing that I’ve always been struck by is that because it’s that first month of pregnancy, at least the data coming out of the cohorts here in California, only about 30% of women were taking a prenatal vitamin in these cohorts in that first month. So there’s a lot of room for improvement, right? Because a lot of them haven’t come for their first prenatal visit yet. Once you get beyond that first or second month, you’re up to 90% of women who are taking the prenatal vitamin. But at that point, yeah, is it more harm than good for the wrong people? Again, it’s a blanket recommendation, where maybe what we should be doing is more precision medicine-based.
Dr. Janine LaSalle: Right? Because there are known genetic polymorphisms, like the MTHFR TT allele, that if you have that—it’s like 15% of the population—they’re the ones that really need it the most because they don’t metabolize folate as well. So, if we could do a screen before to know what folate level to give everyone… But I think there is potentially a harm in both too much and too late because we know the protective window is very small and if you miss it, it might be too late.
Dr. Kara Fitzgerald: Oh, that’s just very interesting. I’m so grateful that you guys are doing this work. We have folate fortification, so there’s exposure there. In non-dairy milks, it’s almost like drinking a multivitamin. There’s always folic acid present and B12. It’s interesting and it makes me think of Randy Jirtle’s work. They made huge differences in the agouti mouse gene expression with those early methyl donor exposures.
Dr. Kara Fitzgerald: All right, where do I want to go? Let’s first talk about mapping out the placental methylome and what that is, and we could layer on the placental-brain axis and specifically some of the main genes you’ve identified, the placenta being a surrogate marker of the brain, the fact that the methylation patterns are almost in reverse to what we think about in adults. Let’s get into that whole story.
Dr. Janine LaSalle: Yeah, I guess you want a little backstory on why we decided to do this. It was interesting because this was around the time when the first maps— I think it was about 2011— when Ryan Lister from Joe Ecker’s lab published some of the first whole genome bisulfite sequencing maps of the human methylome. When we had the human genome from 2000, this was the first time I started looking at these maps and thinking, wow, this is really different and I’d been in epigenetics for a while. One thing that was buried in their paper was this phenomenon called partially methylated domains. It’s like a landscape. If you can think about hills and valleys, if you’re looking across a chromosome and the total levels of methylation, there are these valleys that dip down in the overall methylation level. And they’d seen that in a fetal fibroblast line, IMR-90.
Dr. Janine LaSalle: I was familiar with that paper, and then we had done our first whole genome bisulfite sequencing in a cell line model. It was a neuronal cell line, but it’s a cancer cell line. We were looking at the data, wondering why the methylation was so low. We were mostly interested in the imprinted region of 15q, but we were puzzled by why the overall methylation level was dropping down.
Dr. Janine LaSalle: So, we started thinking about partially methylated domains. Diane Schroeder, my postdoc at the time, suggested trying a tissue with low global methylation, and that was placenta. So we collaborated with folks here to get placentas, and that’s where we basically mapped out that– You know, partially methylated domains had been seen previously in cancer and in this fetal fibroblast line, but in most human tissues, if you look at any cells in the blood or most organs in the body, it’s high methylation overall, and of course it dips down over those CpG islands.
Dr. Janine LaSalle: So it’s part of this weird landscape. That brings us to why is it wacky and what I like to call “opposite world”, because it’s the reverse of what you’ve probably been trained in methylation, which is that methylation is always silencing.
Dr. Janine LaSalle: In the placenta, yes it’s true that if you look at CpG islands, the ones that are repressed have higher methylation than not, so that fits. But when you drop down this whole region, it’s the opposite. So now, gene bodies—it’s like the default is low methylation everywhere and when the gene starts being expressed, you see higher levels of methylation over the gene body.
Dr. Kara Fitzgerald: In the placental methylome, you see higher methylation.
Dr. Janine LaSalle: Higher methylation correlates very nicely with gene expression. It’s almost like gene expression leaves these footprints behind in the landscape. That got us thinking, well, if there are footprints left behind, could we use that to then look back in time? It’s a time capsule of what happened in utero. So that’s how we got into thinking about first, just discovering that the placental methylome is different—it looks more like cancer than it does somatic cells. And then also thinking, could this be used potentially for predicting or uncovering things that happened in utero? So from there, it got us to then thinking about, well, could we use this for the brain?
Dr. Janine LaSalle: In parallel to all that going on, we were also starting to do whole genome bisulfite sequencing in autism brain samples. You don’t get a lot of postmortem brain samples, and there were several studies together that you could see there was definitely an epigenetic signature in the brain. But obviously, for postmortem brain samples there are a lot of reasons why you don’t get much information at all clinically about these samples. And obviously, if you want to try to do any prediction and prevention, you can’t sample a child’s brain. So there was a push in the field to ask, could there be surrogates for the brain? Most people looked at blood and saw some things, but blood was never as convincing.
Dr. Kara Fitzgerald: It’s been a huge struggle to really put our fingers on anything for diagnostics or prediction. I think you’re really kind of the first to come forward with something that could be game-changing. But go ahead.
Dr. Janine LaSalle: Yeah. So then, again, I’m collaborating here. I’ve been fortunate to have some great folks here through our MIND Institute, which focuses on neurodevelopmental disorders, as well as our environmental health focus. We have epidemiologists who started this prospective study, Rebecca Schmidt and Irva Hertz-Picciotto. What they did, is they recruited moms who already have at least one child with autism and are planning their next pregnancy. This way, they can look before the child gets diagnosed with autism because that second child has about a 22% chance of having autism. So it’s a much higher risk. You can think of it as an enriched risk cohort. So we’re looking within the family, because if you did it in the general population, autism is up to 2% of the population now, but it would take a lot to get enough autism samples. And I call this a lesson in patience because we did wait a while.
Dr. Janine LaSalle: It’s not an easy study to do for them. It took ten years of collecting samples before we really had enough to even- We did one study earlier as proof of principle to see that we could do it, and then in 2022 we published the larger study where we had a replication group. Danny Fallin’s group at Hopkins had a similar type study, so we could do a replication with them.
Dr. Kara Fitzgerald: And this was in Genome Biology, I believe you published it, and it’s available as a free, full text. We’ll link to it in the show notes, you guys.
Dr. Janine LaSalle: Yeah, that was exciting because it was a long time coming. In the second paper where we had enough data and could do the replication, we really had a very strong signal on chromosome 22, and it was in this region with no known genes, what I call the off-the-map regions of the human genome.
Dr. Janine LaSalle: It turns out there was a little transcript in there. It hadn’t been characterized- You know, it’s one of those that has an LOC with a bunch of numbers after it, so it’s not well annotated by any means. So we did functional studies to figure out, okay, what is this? it’s been annotated as a non-coding RNA, but we were curious about whether there may be a peptide involved. So my postdoc at the time, Antonio Gomez, was very clever about saying, okay, let’s go in and find the most predictive peptide and design an experiment to look for that. So we were able to show that this gene, which we eventually named NHIP, for neuronal hypoxia-inducible placenta-associated. It turns out that it makes a 20 amino acid peptide. All we know now is that it goes to the nucleus.
Dr. Janine LaSalle: We are currently working on it and we got a new grant to understand its potential. It’s hard when you get something brand new because there are no reagents, so we have to develop all those ourselves. But the promise is that it looks like it’s neuroprotective from the evidence from that paper, because it was lower in autism. The methylation was lower, and the expression was lower in both placenta and the postmortem brain, suggesting that this is something that is induced with oxidative stress or hypoxia during pregnancy. And if you inherit the genetic predisposition to not express as much of it, then that genetic predisposition was also associated with the risk for autism in this small cohort. But obviously, we need to do more to figure out if this is going to be a more general diagnostic or therapy for autism.
Dr. Kara Fitzgerald: And this is where folate would obviously be successful. So you’re identifying this basically unknown gene. It was sort of placed in the junk region? Is that right?
Dr. Janine LaSalle: Well, it’s a very repetitive region of the genome so a lot of times it’s left off the maps. It’s not well represented on either the methylation arrays or the SNP arrays. So yeah, it wouldn’t have probably been seen by other approaches.
Dr. Kara Fitzgerald: Yeah. Minor details when we just dig in. Minor details. So you discovered that this was associated with hypoxia and paradoxically, it’s hypomethylated and therefore inhibited in individuals with higher risk for autism in this cohort that you were looking at.
Dr. Janine LaSalle: Right. And there’s also a genetic– Yeah, I didn’t cover that yet. Because it was such a striking thing, and I thought, well, maybe there’s something genetic going on too, because it’s rare that you see something that large. And so we did find there’s an insertion, a 1.7 kb, an extra chunk on the chromosome upstream of the gene. If the child inherits two copies of that insertion, that’s the risk for autism. We did see some in the typical group that had that, but it was more likely that their mothers took the prenatal vitamin.
Dr. Kara Fitzgerald: Okay, that’s where I was going with that.
Dr. Janine LaSalle: So the numbers are really low, but it suggests that even with the genetic risk, which is pretty common in the population, we’re still figuring it out. But it’s actually in some of these larger databases, and about 70% of people have at least one allele of this insertion, and probably 10 to 15% have two copies. But again, it’s similar to the numbers for the MTHFR genetic polymorphism that I mentioned. So if you take the prenatal vitamin, you can overcome that genetic risk in many cases.
Dr. Kara Fitzgerald: Okay. All right. So how did you guys figure out it had to do with hypoxia?
Dr. Janine LaSalle: Yeah. Well, it was mostly that we were from looking at where it is expressed in other cell lines. What was remarkable is we have this cell line model called LUHMES (Lund human mesencephalic). It’s a neural precursor cell that you can induce to become neuronal. And when we looked at the undifferentiated versus the neuronal differentiation, we saw NHIP increased, so it’s associated with neuronal differentiation. Looking at the gene list we also overexpressed it and were looking at all the genes that were overexpressed, and hypoxia came up in that. So the combination of the neuronal differentiation, which already kind of induces oxidative stress anyhow, because the neuron is changing very rapidly, metabolically. So then we decided to try this hypoxia mimetic, which is cobalt chloride, and interestingly, the differentiated neurons were responsive to that; they became increased reactive oxygen species in response to that. So that’s the question we’re asking: is NHIP maybe protective? Right now, it’s correlated with it. We’re asking if it’s going to be protective.
Dr. Kara Fitzgerald: Right. So actually being able to have it on, doing it’s thing. Because there’s a lower incidence of autism, it’s quite possible that it’s–
Dr. Janine LaSalle: Yeah. And if we could add it back as a peptide, that would be ideal. But it also could be added back as a gene as well.
Dr. Kara Fitzgerald: Say that again. It could be edited?
Dr. Janine LaSalle:– No. Added, like, therapeutically used as a peptide or–
Dr. Kara Fitzgerald: Prescribe a peptide in moms who are pregnant? Or would you do it preconception?
Dr. Janine LaSalle: Yeah, these are all good questions. Yeah. I think we could identify moms at risk and think about this as a potential therapy. It’s down the road. We obviously have more work to do, but –
Dr. Kara Fitzgerald: Yeah. Okay. All right. I’ve got a few questions from this. So, you identified this placental gene and named it NHIP. Is this something that we’ll be able to use to take a placental specimen? What would be the ideal use?
Dr. Janine LaSalle: Yeah. That’s where we’re thinking, potentially, the cell-free fetal DNA. So far, we’ve published on this in a rhesus macaque model, and this is part of a collaborative group where we were looking at the effect of maternal obesity.
Dr. Janine LaSalle: It’s a known risk factor for autism, ranging from about 1.6 to 2-fold higher incidence of autism. For severe obesity, it gets up to about a 2-fold increased risk. This was interesting because we have a primate center here, and a lot of the animals in captivity, like humans, are getting obese, including the rhesus macaques in the colony. So, they took ones that were already higher body weight, obesity in the macaques compared to the lean, and they were actually able to do two different interventions as well. And this was headed by Cheryl Walker, who’s an obstetrician and they did a statin drug versus calorie restriction. The numbers are really low for macaque studies, much lower even than human studies, but it was interesting because we were able to use the methylome to then go and look for changes within these four groups.
Dr. Janine LaSalle: And again, we found some big regions. NHIP was there. The interesting thing about NHIP is that it’s primate-specific. I’ve looked in that locus– Again, it’s not annotated, but I’ve looked in this locus and that one was also showing up in this model. We found two other regions, and since primate genomes are even less well-annotated than humans, we found a region that’s not well-known, although there’s an miRNA there that was interesting, and probably some host genes for that miRNA. And then we found this whole DUX4 group ofgenes that usually work together that were there.
Dr. Janine LaSalle: The interesting thing for the clinical side is that even though this is an animal model, when we took those methylation regions and compared them back to the placentas and the brains that we’ve done before for those epigenetic signatures, there was significant overlap with human autism samples.
Dr. Janine LaSalle: So, a lot of times we go back and forth between human and animal models. We usually like to start with the human and then try to have an animal model that models that human condition, and then we try to bring what we find from the animal model and compare it back to the human signature. And in this case, yeah, we definitely got a strong signature that overlapped with human autism and other neurodevelopmental disorders.
Dr. Kara Fitzgerald: So, NHIP is a piece, but you’re expanding it to other regions?
Dr. Janine LaSalle: Yes. I guess I got off the track of the cell-free DNA. The story behind that is they were trying to… The thing about monkeys is it’s really hard to get their placentas at birth like we would with humans because it’s important to leave them alone during the birth process to ensure they don’t get separated from their infants. Otherwise they might reject the infant later.
Dr. Janine LaSalle: So we couldn’t get the term placenta. Cheryl had done the equivalent of CVS for the monkeys to get placental biopsies, but for some reason, it’s a stringy tissue, and it was really hard to get DNA out of that. But what they did take was blood throughout the pregnancy at four time points. They were already doing cell-free DNA because they only wanted male births. Again, monkey studies are really expensive and hard, so they only wanted males so they were screening for Y-chromosome DNA to confirm that they were male births. We were able to get that cell-free DNA, which includes the fetal cell-free DNA and that’s where we really saw these patterns. It’s the cell-free DNA from that.
Dr. Janine LaSalle: And what was remarkable, actually, is these regions that we found in the cell-free DNA, often very much matched what we saw in the brain. So, it’s like the placenta cell-free DNA was predictive of the brain. We also had behavioral– The nice thing about the monkeys is that you can then follow them up and do behavior, so there was at least, the social behavior they were measuring was correlated then with –
Dr. Kara Fitzgerald: The genes you were seeing. What you were picking up. So cell-free DNA will have some fetal DNA, and you can differentiate that in the laboratory. And that would be a much easier collection than a placental biopsy. Clearly.
Dr. Janine LaSalle:Yes. If you want to be able to tell during pregnancy what the risk is then I think that’s an interesting idea.
Dr. Kara Fitzgerald: Where is that in development? When are clinicians going to have access to that?
Dr. Janine LaSalle: Yeah. Well, we are doing this now, going back to this MARBLES study, which I was telling you about, the prospective study, we’re going back to do cell-free fetal sample on the ones we had placentas from, so we have that in progress. I’m a little wary of pushing this too much because we also have the neurodiversity community that will say, wait a minute. You’re trying to… Yeah. I don’t want to… I mean, wary of what’s happened with Down’s syndrome and the genetic test. So I think it needs to be clear that this is not a genetic test and it probably will never be 100% predictive. But if we could do an intervention during pregnancy to help the maternal environment. But again, that’s the question. I think we need to have a clinically actionable treatment for it before–
Dr. Kara Fitzgerald: Well clearly, number one, as you’ve already said, would be adequate methylation support, or as you guys are continuing to investigate whether in some cases there might be too much. But the other thing that you have looked at in your work are toxin exposures.
Dr. Kara Fitzgerald: In fact, you talk about this methylation, this very unique placental landscape, these imprints – maybe imprint is not the best word – but these methylation signatures are like reading the book through time, like seeing the environmental exposures over the life of the placenta. So it’s what the fetus has been exposed to. And I might be able to say that toxins are influencing this landscape, correct?
Dr. Janine LaSalle: Yes. Yeah. And so we’ve looked primarily so far at PCBs. So these are chemicals that were used starting in the ’30s, ’40s, ’50s. They were banned in the mid-’70s, but they’re called persistent organic pollutants for a reason. They don’t break down very easily in the environment. They are lipophilic, so they bind to lipids and they bioaccumulate up the food chain. And of course, humans are at the top of the food chain.
Dr. Janine LaSalle: So although there’s a decline in the environment over time,
and actually, in our study, we were using the MARBLES cohort again, we could see one of the best predictors of the amount of PCB in the maternal blood was the combination of their age and year of birth. So it’s decreasing, but then when we correlated the PCB levels to the methylation levels in the placenta, what we ended up finding were some of these partially methylated domains over genes that were also observed in autism. So the CSMD1 was one that really came out, AUTS2, which is another autism gene, those were two that came out. And interestingly, we also were collaborating with another group, Pam Lein’s lab, who is a toxicologist, and she developed– So again, we go from the human and we then want to model it in a mouse. So what she did was design a mouse model of PCB exposures based on the levels and the combination. So there’s multiple congeners of PCBs. It’s a different mixture.
Dr. Janine LaSalle: So they took the same mixture of PCBs that were found in the MARBLES cohort, which is a high-risk autism cohort, so moms that have a high risk of having a child with autism, we model that in the mouse, and then we look at the placenta and brain in mouse. And remarkably, those two genes came up with the same thing, the CSMD1 and AUTS2. So it gives you some confidence that we are finding, sort of, these epigenetically susceptible genes that when we look across species, we still see them.
Dr. Kara Fitzgerald: I have two questions looking at this through the functional medicine lens. So early in my career, I was in a clinical laboratory, and we were looking at the methylation cycle. We were looking at glutathione and the amino acids involved in that and s-adenosyl-methionine and s-adenosyl-homocysteine, and oxidative stress, like eight-hydroxy two-deoxyguanosine, and lipid peroxidation and so on and so forth.
Dr. Kara Fitzgerald: And you know what was interesting? I did this itty bitty data dive, looking at a cohort of kids with autism and their homocysteine status, just as homocysteine being sort of the surrogate marker of what’s happening with methylation, and it was low compared to what we anticipated in a neurotypical cohort. That was pretty routine back when I was in the lab, that you would see imbalances in methylation and sulfuration and glutathione status, sort of allowing oxidative stress to be kind of uncontrolled or insufficiently controlled. And that was demonstrated and published on by Jill James. It’s really interesting.
Dr. Janine LaSalle: Jill James, yeah. I was going to say. She’s done quite a lot on that. Yeah, yeah.
Dr. Kara Fitzgerald: And does that fit with what you’re talking about and perhaps why a prenatal, I mean, just thinking beyond folate. Folate is a piece of it, but B-12 is there, and maybe there’s a little bit of choline because it’s prenatal. But anyway, I’m just curious.
Dr. Janine LaSalle: Yeah. I think the prenatal vitamins are counteracting multiple exposures and multiple genetic contributions too, which may not be 100% penetrant if you actually had proper nutrition as well. So, yeah.
Dr. Kara Fitzgerald: And offsetting the PCB damage and the myriad other toxins that you’ll uncover, I’m sure, over time.
Dr. Janine LaSalle: Yeah. And we are looking at that in the mouse model of the PCB exposure to look at folate as a potential protector of that.
Dr. Kara Fitzgerald: That’s fascinating. Back then, you know, this was early in my career, it looked like the incidence of autism was just rising meteorically, from very low incidence in the 80s to I think we must be what? Probably 1 in 5 boys now or something really pretty high at this point.
Dr. Janine LaSalle: Yeah, its’ 1 in 5 boys now, according to the CDC. Yes.
Dr. Kara Fitzgerald: And so why? Why is that? What do you think?
Dr. Janine LaSalle: It’s a very difficult question to answer. But I agree with you. It was interesting because I wasn’t necessarily working on autism. I came here in the late 90s, and that’s about the time the Mind Institute started in Sacramento and so I was going to a lot of the talks. And what’s nice is there were a lot of different disciplines coming together to think about autism. I remember seeing these numbers going up at that time and the epidemiologists would say, well, we can’t know because the diagnosis has changed. Every time a new DSM (Diagnostic and Statistical Manual of Mental Disorders) comes out, the diagnosis changes again.
Dr. Janine LaSalle: So you really can’t go backwards and ask the same question because the diagnosis– And that’s really, I guess, been one of the motivating factors for us too, is can we come up with a molecular test or at least something else to go along with just the behavioral diagnosis, because there are biases. If you look over time, girls were really underdiagnosed previously. It’s getting better, but they’re still probably underdiagnosed compared to boys. So, I do think that there’s a biological reason why there’s more boys, and it’s about a 3 to 1 ratio. We actually are looking now at this historical cohort. Well, what we did is go back from blood spots to– I don’t know if I have time to get into that cohort, but another cohort where we could go from newborn blood spots from kids that had been diagnosed and their years of birth starting from about 1989 to 2014. And we can see even in that data set, there’s almost a different epigenetic signature between the two.
Dr. Janine LaSalle: I probably shouldn’t talk about this too much because the paper is close to coming out. So I guess I just say that we are seeing it in the data that the diagnosis has changed over time, and it makes it very difficult to say that autism diagnoses from different decades are the same thing biologically. And when you think about across the world, how do we even know the real prevalence of autism in other countries if the diagnosis– You know, a lot of kids may not be getting diagnosed. There’s a lot of health disparities in that space too, if you want me to get into that. But I mean, you can see it again in the California data that the kids with autism are more likely to be sort of higher socioeconomic, compared to the ones that would be maybe diagnosed with developmental disorders disability, DD instead of ASD, and you can see that in the data. So it’s frustrating as a researcher because–
Dr. Kara Fitzgerald: Just lack of a precision diagnosis.
Dr. Janine LaSalle: Lack of precision. Yes. But I mean, that’s probably true of a lot of diagnoses in medicine, but I think it’s something everyone needs to face. And I think researchers especially need to be aware that just because your sample says they have autism, you really do need to pay attention to what the criteria for diagnoses were and when it was done, because it’s changing over time.
Dr. Kara Fitzgerald: Right. Right, right. Yeah. So certainly having a molecular diagnostic will move things forward.
Dr. Janine LaSalle: Yeah. What I like about the prospective study we’ve done is that all of those kids were diagnosed at the same place. They’re all done at the Mind Institute. And they go through the typical kids too. And what’s interesting, is that roughly 22% have autism, but only about 50% are completely typical on all measures, and there’s this other category that we call non-typical development that is somewhere in between.
Dr. Janine LaSalle: And they’re the ones that have learning difficulties or probably ADHD. It’s kind of that other category. So again, the whole spectrum of neurodevelopmental disorders is larger than just autism. And again, it seems that the higher risk for autism goes along with higher risk for these other disorders as well.
Dr. Kara Fitzgerald: It’ll be interesting as you peel that back over the years what you’re seeing with these molecular signatures.
Dr. Janine LaSalle: Yes. Yeah, I know because I think we might be able to get at some of the disparities and, you–
Dr. Kara Fitzgerald: Yes,and concurrently, one of the reasons physicians migrate to functional medicine is because we’re so moved by having a sense of what the mechanism is because that can inform our clinical decision making. Just the name of the hypoxia-inducible gene suggests the solution might be supporting antioxidant status, and just linking that back to what we know about glutathione.
Dr. Kara Fitzgerald:And that just walks hand in hand with the fact that we’ve got a toxic burden, all of us. It’s unmistakable. And it does seem to be increasing. And to your point, the persistent organic ones are hanging around, plus we’re layering on additional–
Dr. Janine LaSalle: Yeah. Well now we have PFAS, (Per- and polyfluoroalkyl substances), which is another chemical, and yeah. We have a group here at Davis who’s very interested in wildfire smoke exposure because we’re in California. And, we just had some really bad years of wildfire smoke exposure and especially in pregnancy, it’s probably all the combination of things we know are bad about air pollution, about smoking. But, yeah, you get all these chemicals delivered through the smoke.
Dr. Kara Fitzgerald: And some of the pretty scary stuff around glyphosate and some of the cancer and neurological changes. All right, let’s see what else I want to ask you here.
Dr. Kara Fitzgerald: I just want to go back to – I know there’s a couple questions and one, hopefully I’ll pull back here in a second – but just thinking about the placental methylome. I want to nail down on why the hypermethylation signature is suggestive, not of active gene transcription, but of gene transcription in the past. I mean, can you just speak to why does the placenta methylome A) Look like cancer and, you know, B)…
Dr. Janine LaSalle: It’s a lot to unpack. Let me think.
Dr. Kara Fitzgerald: Well, let me say this. People listening, we know tumor suppressor gene hypermethylation is a thing in cancer. So there’s an understanding of that here and that the oncogenes can actually be on. So there’s this paradoxical occurrence in the tumor microenvironment of hypermethylation of protective genes, hypomethylation of damaging genes in the cancer microenvironment.
Dr. Kara Fitzgerald: Incidentally, that’s in the aging methylome as well. But then you’re seeing it in the placental methylome, so I want to understand that. And then layer on why hypermethylation is suggestive of activation. Because anybody who’s been listening to this podcast and following our conversations here on epigenetics, you know, I tell everybody to just imagine those red lollipops, right? And they inhibit transcription. Just structurally you can’t get in there. The red lollipops being the CPGs on the cytosine and you can’t dock. So when I was reading your work, I was completely struck by that because–
Dr. Janine LaSalle: I know. I literally had to go back and change up my teaching slides after I saw the whole methylome landscape. And I’m like, you know, this is not that simple anymore.
Dr. Kara Fitzgerald: And, well, and you know what? There’s hints of that in the literature so I would read in the literature and there’s like a sentence of, you know–
Dr. Janine LaSalle: Even back in the days before Prader-Willi locus, when we were doing these things, honestly I did southern blots to look at DNA methylation in those days, and there were ones that were the opposite, right? We definitely had the one at the imprinted control region, which was always the maternal. But then you had these other regions that were the opposite. And now that we have the whole view, we see it. And so this is a pattern– I’ll get back to the placenta and cancer, but let me just cover this first because you see it on the inactive X chromosome and you see it on imprinted regions, where there’s this big block of heterochromatin on one–
Dr. Kara Fitzgerald: Can you define that? Define imprinted regions please. Yes.
Dr. Janine LaSalle: Yeah. So imprinted regions are where there’s a difference between what’s inherited from the maternal and paternal chromosome. So for instance, in Prader-Willi and Angelman syndrome, you can have the same deletion on chromosome 15q11-13, but Prader-Willi syndrome would have that deletion inherited from the paternal and Angelman would have it deleted from the maternal. And it’s because there are differences in gene expression across these regions. But it’s this whole block of a chromosome that has an imprint. And like the inactive X chromosome is like that in females, that in every cell you’ve got one of those cells that’s completely turned off and it turns into this block of heterochromatin called a Barr body. So, there are two examples of these big blocks where one of your two alleles is getting turned off as a whole chromosome. And when you see that, those regions actually have lower methylation, and that’s been seen for the inactive X chromosome, and we’ve seen it for sure in the imprinted regions. So it’s acting like that partially methylated domain. So I really think it’s the lack of accessibility to the DNA methyltransferases, which is why you’re not getting the methylation in those regions.
Dr. Janine LaSalle: But again, going back to where else do we see this. In the placenta, yes. But I’m going to go back even earlier before the placenta, because we also did this to ask, okay, why is it in the placenta? And if you go back to the oocyte, the oocyte actually looks like this as well. And it’s interesting, you have the immature oocyte and when it becomes mature, there’s a whole gain of methylation. It increases methylation, but it always increases it over gene bodies. So the placenta is really more like the oocyte and there’s not a lot of data in humans, you can get some blastocyst cultures, and the same thing there. You see partially methylated domains in the oocyte, those pre-implantation embryos, and then once it’s implanted, you think about– So the trophectoderm is that layer of cells, that if you see a picture of a blastocyst at implantation, you’ve got the inner cell mass which becomes the embryo.
Dr. Janine LaSalle: You get that circle of cells around it that is the trophectoderm. The trophectoderm becomes the placenta and the inner cell mass becomes the embryo. So the trophectoderm keeps that pattern that was there in early preimplantation life, whereas the embryo itself gets highly methylated right after implantation. So why does cancer– I mean, I would flip the question, I guess. Because I think we shouldn’t be asking, why does the placenta look like cancer? We should be asking why does cancer look like placenta? Why does aging look like placenta? I think they’re developmental origins, and it’s going back– I mean, why create a new program if you can go back and use a program that you’ve used? It’s erasing some of those somatic marks and going back to an earlier stage. I mean, that’s one possibility. The other possibility is if it really is developmental origins of something like cancer, maybe because of the stress of pregnancy, it didn’t quite finish the program and all the cells and all we take is like a couple of cells in there later on because they have that earlier epigenome marks, they’re more likely to turn into a cancer.
Dr. Kara Fitzgerald: That’s pretty fascinating. Yeah. So in cancer, it could be regressing to a more primitive, less controlled state.
Dr. Janine LaSalle: Right. And primitive is interesting, too, because if you think about it all mammals have pretty high levels of methylation. But if you go to vertebrates before mammals, they actually look more like the placenta in that they have overall lower levels of methylation. And when you do see methylation, it tends to be over gene bodies. So like zebrafish, Xenopus, organisms like that have methylation patterns that look more like the pre-implantation and placental state. So it’s going back not only to an earlier developmental state, but also, you know–
Dr. Kara Fitzgerald: That’s pretty interesting. Okay.
Dr. Janine LaSalle: Primitive state, as you call it. Yes.
Dr. Kara Fitzgerald: And aging looks like this as well. And why is that?
Dr. Janine LaSalle: Okay, unpack aging. Yeah. I mean there’s definitely strong epigenetic signatures of aging. I mean, that’s clear. And they’re different from what you see at telomeres, so is it the result of all accumulated oxidative stress over time? Are we overall eroding some of those programs that we put on and going back to that earlier stage? Yeah, I think these are all really fascinating questions. I haven’t really got–
Dr. Kara Fitzgerald: It’s not your area.
Dr. Janine LaSalle: But no, I follow it. It’s fascinating, but I think a lot of it can come back to those developmental origins. We tend to think that any disease that occurs later on, you think, oh, it must be something I did in recent life. But sometimes it goes back to in utero or potentially even grandparents.
Dr. Kara Fitzgerald: Yeah.
Dr. Janine LaSalle: Think about generational impacts.
Dr. Kara Fitzgerald: Yes. That’s right. Fetal origins of disease. Or, you know, things like the Overkalix cohort or Dutch Hunger Winter where we can see generational influences on the methylome from those exposures, maybe starvation or insufficient food or excess, and then you see that handed down in the development of certain diseases, like cardiovascular disease and diabetes. Sinclair’s lab – and I know we’re at time, but I just want to ask you – David Sinclair at Harvard, and they’re looking at the aging phenomenon in mice in age-related optic neuropathy, and just aging in general.
Dr. Kara Fitzgerald: And he describes it as being able to drive age forward and then kind of reverse it, that a youthful pattern actually remains at some level in the epigenome, even as those aging changes happen. And that seems to be in keeping with what you said. And then I don’t know if you’re familiar with somebody who podcasted with me recently and was again, another tour de force podcast, was Vittorio Sebastiano out of Stanford. He developed a clock. He’s doing cellular regeneration. So they’re using Yamanaka factors to really turn back the hands of time on cells. And then he developed an epigenetic bio-age clock that incorporates the entire methylome and finds that the changes that really radically alter all of the hallmarks of aging are measured on the polycomb genes, I think. So can you speak to that? And define polycomb, too.
Dr. Janine LaSalle: Yeah. So polycomb regions are ones that tend to be developmental transcription factors. But again, they’re part of that landscape of the methylome. You can see them because they always drop down– they’re kind of similar to partially methylated domains, but they tend to be smaller and instead of partially methylated domains being very tissue-specific, like when you do have different tissues, whereas the polycombs are in every tissue you look at you see this drop down. And what’s interesting too, genomically, they have what we call archipelagos of CPG islands, because instead of having one gene with one CPG island, it’s almost a cluster of higher CG content. Which again, I think evolutionarily that’s interesting because they are more, again, leftovers of earlier evolution. Mammals basically have gotten rid of a lot of CG sites because the expense of having a genome with high methylation is that you lose CPG sites. And that’s why we have CPG islands in the mammalian genome.
Dr. Janine LaSalle: But these polycomb regions are almost like remnants of past evolution. They’re very well conserved. So these are developmental transcription factors that we’ve had that are probably the most conserved genes in our genome across organisms. And yeah, their epigenetics is different. They don’t use methylation in the same way. They tend to use the polycomb modifications on histones, so H3K27 trimethylation, that’s where you get these bivalency of histone marks. You can have both an active and an inactive mark in the same region. And it’s really interesting because we think in placenta in these regions that have lower methylation, they’re actually using more of the polycomb marks compared to later on. And that’s also again, shared similarities with cancer where it’s using the polycomb – this bivalency It’s a poised state where the gene’s trying to decide, should I be on, or should I be off. But it can go either way.
Dr. Janine LaSalle: And I think it’s the plasticity of that state which contributes to the plasticity of cancer, the plasticity of placenta. And I did want to say too, placenta is like a cancer. Right. So what it’s doing is going out and grabbing those blood vessels. It’s doing angiogenesis. It’s bringing in blood supply to the fetus. So it’s functionally very similar to cancer.
Dr. Kara Fitzgerald: It’s really extraordinary. It seems like we’re at the precipice – although we’ll no doubt be here for a long time – really penetrating some serious mysteries linking aging to the earliest developmental happenings and from an evolutionary thread pulling that forward. It’s absolutely mind-blowing. And I just again, appreciate you for being willing to spend some time with us. What I was going to say earlier that I was trying to pull out of my brain, is that we’re going to, Dr. LaSalle provided me with a really lovely bibliography, and I know certainly a number of these are free, full texts.
Dr. Kara Fitzgerald: So, if people want to look at the PCBs research that she’s done, the Retts, the NHIP gene discovery journey, and folate turning that on… All of those papers, links will be in our show notes to her full bibliography. Maybe we’ll pop the Jirtle Imprintome conversation in there so that you can refresh your mind on what that’s about. And we’ll put the Sebastiano conversation in there as well. Maybe even Sinclair. I podcasted with him quite a while ago when they were just starting to work on this, so that if you want to do an epigenetic deep dive, we’ll make that available to you in our show notes. I was going to ask you about sex differences. We touched on that a little bit, but we’ll park her bibliography there. I’ll just continue to follow you as some of these things come forward.
Dr. Kara Fitzgerald: The peptide that you talked about possibly being able to be prescribed early in pregnancy, and turning around some of the development of these neurological disorders, and then your continued work in collaboration, looking at toxins and looking at vitamins and so forth. Thanks again so much. And if there’s anything I missed that you wanted to jump in.
Dr. Janine LaSalle: You covered a lot.
Dr. Kara Fitzgerald: We covered a lot. Yeah. Janine, thanks, thanks, thanks for joining me on New Frontiers.
Dr. Janine LaSalle: Okay. Thanks so much.
Professor, Medical Microbiology and Immunology, School of Medicine
University of California, Davis
The LaSalle Lab at University of California, Davis https://mmi-lab.ucdavis.edu/
(530) 754-7598
Dr. LaSalle is a Professor of Microbiology and Immunology at UC Davis with expertise in epigenetic mechanisms at the interface of genes and environment. Dr. LaSalle serves as the Deputy Director of the UC Davis EHSCC and Co-Director of the Perinatal Origins of Disparities Center. Her laboratory uses genomic and epigenomic technologies to investigate DNA methylation signatures in autism spectrum disorders and beyond. Her lab utilizes perinatal tissues such as placenta, cord blood, newborn blood spots, and cell free fetal DNA for epigenomic prediction and prevention of health disparities.
Perinatal Origins of Disparities Center
Environmental Health Sciences Center
Article: Too Much Folate in Pregnant Women Increases Risk for Autism, Study Suggests
Study: Markers of Autism Risk in Babies (MARBLES)
Study: Transposable elements: targets for early nutritional effects on epigenetic gene regulation
On The Placental-Brain Connection
Placental methylome reveals a 22q13.33 brain regulatory gene locus associated with autism
On The Womb’s Strange Epigenome
On Convergent Epigenetic Alterations In Autism Spectrum Disorders
On Circadian rhythmicity and imprinting dynamics of the Prader-Willi/Angenman syndrome 15q11-q13 locus
A Prader-Willi locus lncRNA cloud modulates diurnal genes and energy expenditure.
Snord116-dependent diurnal rhythm of DNA methylation in mouse cortex.
Rett syndrome and MeCP2, focused on sex differences in an X-linked dominant disorder
MeCP2 isoform e1 mutant mice recapitulate motor and metabolic phenotypes of Rett syndrome.
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