Folate & Autism, Unpacked
Prep for the upcoming HHS report
If you had told me when I started doing my PhD in one-carbon metabolism — a metabolic network in our cells that nutrients like folate, vit B12, and choline are important players in — that this knowledge would helpful in unpacking modern contentious debates about prenatal vitamins and speculation-based HHS health policy, I might have picked a different topic. Alas, folate is all the rage nowadays, from the prenatal vitamin industry removing folic acid for purportedly superior methylfolates (to be unpacked in a future post) and now reporting stating that HHS will link ‘folate deficiency’ to autism spectrum disorder (ASD) and highlight the potential of a specific form of folate, folinic acid, as a treatment for ASD. Despite the limited information, this announcement is already getting talked about a lot on social media, and concerning causing worry amongst communities of parents who are having to add another type of folate into the prenatal folate wars, so let’s dive in.
Folate 101
To understand this issue, you need to understand a bit about folate. Folate is an essential nutrient (vitamin B9) but its not one thing - folate is an Umbrella Term that refers to chemical compounds similar in structure that can fulfill the biological roles of folate in our bodies. There are several forms of folate found in the diet that reflect the forms of folate found in the cells of plants and animals, as well as the folic acid that is fortified in our foods and whichever supplemental folate form that is being consumed. The forms that we eat in food/supplements get digested, absorbed, and metabolized by our own cells to other forms of folate that are capable of playing the essential biological roles of folate in our cells. (This is the gist of it - more nuance coming).
Folate Biology
As you can see in the figure above, most folates have what we call a ‘one-carbon substituent’ I.e., they carry a type of single carbon containing unit. Different folates will carry different single carbon units to donate for the synthesis of other molecules.
If you’re deep in this world already, you have probably heard a bit about this e.g., ‘methylfolates’ are commonly discussed - that term ‘methylfolate’ is referring to the fact that the folate molecule can carry a Methyl group at its Nitrogen-5 position in its bicyclic pteridine nucleus on the left side of the molecule. This is a major role of folate in our cells- to carry that methyl group and in conjunction with vitamin B12, remethylate a molecule called homocysteine back to methionine. Methionine can then become activated by an adenosine group to S-adenosylmethionione (SAMe) which is used by dozens of enzymes called methyltransferases that take that methyl group from SAMe and add it to a substrate compound to create some useful product. There are countless products, too many to list, but include the synthesis of phospholipids, neurotransmitters, intermediates in creatine synthesis, breakdown of xenobiotic compounds, etc. DNA, RNA and proteins can also be methylated and this influences the regulation of gene expression (more on this and ‘epigenetics’ below).
Folate’s other major role in the cell is in carrying different types of one carbon units required to make 3 of the 4 bases of DNA. An intracellular form of folate called 5,10 methylenetetrahydrofolate (5,10-mTHF) is required to synthesize the T in DNA and 10 formyltetrahydrofolate (10-fTHF) is required to make the A and G.
These roles of folate are why it is important during all stages of life - your body needs to undertake those methylation reactions all the time and the body is always making new DNA to support cell maintenance and turnover. This is why folate is considered an essential nutrient and if you deprive folate from the diet, you get characteristic deficiency symptoms. The major symptom of folate deficiency is a megaloblastic anemia, wherein red blood cells are immature and present with an enlarged phenotype (hence the megalo-) due to reduced DNA synthesis and an inability to divide. Many other symptoms of folate deficiency can present, particularly if deficiency is prolonged, including fatigue, depression, glossitis and weight loss. Biochemically, with generalized folate deficiency due to low intakes and/or impaired absorption, serum levels of folate will be low and homocysteine will be high, reflecting impaired capacity to remethylate homocysteine to methionine. Folate deficiency is, however, pretty rare in the USA population, due to generally diverse diets being available and the mandatory fortification of grains (for enriched grains with standards of identity) with folic acid in the food supply.
Folate and Development
While folate deficiency is quite rare in the broader population, it’s still an area of intense study, particularly as it relates to development. Development is a time period when cells are rapidly growing and dividing, in need of new DNA to be synthesized. Development is also a time period when cells are establishing their own cellular identity - all cells have the same DNA but cells of different tissues function quite differently and this is due to how they regulate their gene expression. That regulation of gene expression that determines cellular identity refers to the field of ‘epigenetics’. When humans are developing and their cells are establishing their genomic identities, they are considered to be more sensitive to environmental factors that might influence the establishment of that identity. At a cellular level, part of that identity is the methylation of DNA, as well as the histone proteins that DNA is wrapped around -these methylation marks impact how accessible DNA is to protein complexes that regulate the transcription of DNA and subsequently how proteins that govern cell function are made. Given folate’s central role in providing methyl groups, there’s a lot of interest in how modifying the availability of folates throughout development might impact how the epigenetic code is esetablished and subsequently impact health and disease risk. It should be noted that much of the epigenetic code is determined by the cells own programming and only some sites for DNA and histone methylation appear particularly sensitive to the supply of nutrients - this remains an active area of research that doesn’t really inform our clinical recommendations around folate (although many naturopaths online will have you pretending it does) but it is nevertheless part of the underlying biological rationale for researchers who study how folate availability at key developmental time periods impacts tissue function and health/disease risk.
Folate and Neurodevelopment
Folate’s role in supporting cell proliferation and methylation led folks pretty early on to study folate and the brain development, with seminal findings indicating the brain’s sensitivity to folate supply:
In the 1950s, rat models of folate deficiency using antifolate compounds reported reductions in brain weights of pups born to exposed dams during the latter half of pregnancy (note: rodents eat their poop that contains folates, making the induction of dietary folate deficiency difficult and requiring chemical methods to model a folate deficiency). In addition to low brain weights, the offspring rats also had high rates of cleft palates, tissue that develops out of the neural crest tissues, and are a type of birth defects in humans that folic acid likely helps to prevent.
In the early 1960s, the first 3 patients with congenital folate malabsorption were identified, and had not only the expected megaloblastic anemia but also had severe mental retardation, ataxia, and convulsions (since they had impaired folate absorption during early life when the brain is rapidly developing, this was strong evidence that severe folate deficiency can impact the brain’s development).
Folate’s shining role in neurodevelopment broke through in 1960s-1990s with the study of neural tube defects (NTD) and the ultimate link between folic acid supplementation and NTD prevention. Neural tube defects refer to diagnoses like spina bifida and anencephaly that result from impairments in the develop of the neural tube, the early developing tissue that gives rise to the brain and spinal cord.
NTDs were a major area of study in the 1960s-1980s, particularly in the UK where rates were high at a population level but variable between regions and associated with class. Nutrition was an early consideration as a factor that might predispose to NTD risk, with some but not all studies finding differences in folate status and risk of NTDs.
The interest in folate and other vitamins led to clinical trials, with a seminal 1991 multi-centre trial demonstrating a strong benefit of folic acid alone or alongside other micronutrients in preventing NTD recurrence in pregnancies compromised by a previous NTD. Subsequent research would ultimately lead to broad recommendations for women of childbearing age to supplement with folic acid to prevent the risk of NTDs and ultimately for population-wide folic acid fortification of the food supply in 1998 - both strategies aim to ensure that folic acid intake and folate status are adequate for individuals when they become pregnant, given the development and closure of the neural tube in the first month of pregnancy before more are aware of the pregnancy.
Folate and NTDs continue to be a hot area of research in neurodevelopment. There are many questions that remain about the exact mechanism of how folic acid prevents NTDs and what factors predispose to risk. Its notable that only a small fraction of the population appears susceptible to a NTD - it does not appear, like many state, that NTDs are a result of ‘folate deficiency.’ Many pregnancies complicated by NTDs documented in the literature are not necessarily abjectly folate deficient (I.e., presenting with megaloblastic anemia) and not all even exhibit lower serum folate levels (levels are typically moderately reduced but not drastically deficient). Similarly, you can’t induce NTDs in rodent models, without further compromising the one-carbon metabolism genetic network (I.e., you have to knockout a gene in one carbon metabolism to sensitize to developing a NTD). Folic acid supplementation looks to be preventing a subset of NTDs in a subset of individuals who are susceptible, possibly through genetic risk that compromises one carbon metabolism. I bring this up not to complicate things or as a fun nuance of NTDs but rather, because a signal for folate in neurodevelopmental outcomes without a definitive mechanism and with incomplete penetrance will visit us again shortly when we think about autism.
Folate and Autism
Autism hit the larger public consciousness in the USA in the late 1990s, following controversy around Wakefield’s 1998 now-retracted fraudulent paper claiming a link between the MMR vaccine and autism. Congress responded passing the Children’s Health Act of 2000 that required the expansion of autism research and surveillance. As increased surveillance has led to significant increases in diagnosis and an appearance of increasing incidence, we’ve inevitably seen folks focus on environmental factors that could contribute to autism rates. In retrospect, it’s not too surprising that folate would be something studied in relationship to autism. Folate was hot on the scene as a nutrient impacting neurodevelopment and it was added into the food supply with fortification in 1998 - it’s never good to have your public health intervention with a ‘synthetic’ vitamin begin just before a large screening campaign that captures predominantly mild autism diagnoses.
At a high level, the evidence above is pretty convincing folate intake or status of an individual can impact neurodevelopment. The question becomes, is folate at all related to neurodevelopmental conditions like autism? Per the coming HHS report, the answer from the government’s perpsective sounds like it will be ‘yes’. But we should pause here for a second because there are various ways that the high level question of ‘is folate related to autism’ has played out in the research, that may or may not be adequately communicated in whatever HHS puts out and in the media fanfare to follow. The folate-autism hypotheses that exist look like the following:
A general folate deficiency hypothesis during pregnancy - A lot of the research, including much of what has made headlines over the past decade, has asked whether low serum folate or self-reported use of folic acid supplements during pregnancy is associated with the risk of ASD in offspring. This is a reasonable hypothesis to test (i.e., does low serum folate or inadequate folate supplementation increase ASD risk) = unfortunately the literature on this topic is quite weak. In an ideal world, we would have cohorts designed to ask this question that assessed folate status and supplement use multiple times (pre-pregnancy, throughout pregnancy) and followed up infants who were assessed for ASD diagnosis, severity of symptoms etc. Unfortunately most of the literature is convenience samples measuring a single time point of serum folate and querying folic acid supplement use, and relies on ASD diagnosis alone to assess risk (an issue given that not having a documented diagnosis doesn’t necessarily mean individuals wouldn’t qualify for it). The relationships for self-reported folic acid supplement use tend to find protective effects (but this is not always the case), and there is concern about excessive supplementation. Similarly, folate status measures in better cohorts suggest a U-shaped curve between serum folate with both low and high levels increasing risk. The high levels of folate that are associated with increased risk reportedly reflect high levels of unmetabolized folic acid, suggestive of excessive supplementation and/or an underlying genetic architecture of risk (e.g., Dihydrofolate reductase variants that limited the capacity to reduce supplemental folic acid to enter the folate cycle) — but there’s only a single cohort assessing this. The good news on both fronts is that the available data support existing diet and supplementation recommendations, but the jury is still out on whether there is a generalized ‘low’ folate status that we can say causally impacts the risk of ASD.
A general folate deficiency in ASD hypothesis - Another piece of the literature has examined folate status indicators in largely case-control studies comparing individuals with ASD to matched controls. The studies have identified a somewhat consistent trend for low folate status indicators, as well as other markers like oxidative stress, although few studies are conducted similarly enough to meta-analyze the data and come up with a meaningful conclusion. There’s no consensus on how widespread this phenotype is, whether it relates to ASD etiology or is just downstream of behavioral differences (i.e., higher food selectivity) that impact in dietary intake. There’s not enough metabolic data to say there’s something about ASD that increases the folate requirements or shows a benefit in behavioral symptoms from high dose folate supplementation in routine ASD care at present. Relatedly, very small, underpowered studies indicate that individuals with ASD may have slightly higher prevalences of common genetic variants in one-carbon metabolism but again, these are uncertain estimates and these genotypes are commonly found in many individuals without ASD - there’s no clear benefit to genotype in assessing risk or guiding treatment at present, although many will use genotype to guess at folate dose and form.
A cerebral folate deficiency hypothesis - This hypothesis is (IMO) the one most worth delving into and where the biology is most interesting, although the clinical implications are extremely preliminary. Given the HHS call out to treat patients with folinic acid, its worth diving a bit deeper into the cerebral folate deficiency hypothesis.
While folate and NTD research was all the rage, researchers identified a rare condition of folate deficiency specifically in the brain in humans, first in a patient in 1994, followed by 25 more cases in 2002 and 2004 papers. These patients with ‘Cerebral Folate Deficiency’ (CFD) exhibited normal serum/red blood cell folate levels as well as homocysteine and were without anemia (indicating they didn’t meet the definition of a classic/systemic ‘folate deficiency’) but had uniquely low folate (5-mTHF) concentrations in the cerebrospinal fluid (indicative of a ‘tissue-specific’ nutrient deficiency). This low CSF folate correlated with an array of symptom starting age of 4 to 6 months that progress out to 6+ years- cognitive impairments, visual disturbances and hearing loss, movement disorders, sleep disturbances, and atypical behaviors. CFD is another landmark piece of evidence showing just how impactful inadequate folate during brain development can be.
CFD didn’t appear to be genetic from these seminal descriptions (later studies would find genetic causes) - sequencing of the major choroid plexus folate transporter, the folate receptor alpha, that transports folate from the peripheral blood into the brain and cerebrospinal fluid (CSF), appeared normal in the patients. CFD also didn’t appear to run in immediate families. Rather than a genetic cause, in a 2005 landmark paper, patients with CFD were shown to have defective folate transport secondary to autoantibodies in their sera that bound to the folate receptor, blocking folate uptake into the brain. Interestingly, a few years prior, mice genetically engineered to lack this receptor in the whole body showed defective embryonic development, including neuronal development, that could be rescued by high dose folinic acid. Researchers working with CFD patients jumped on very high dose folinic acid treatment (10-100X compared to dietary intakes; 10s of milligrams, as opposed to 100s of micrograms normally consumed in the diet) pretty early on as a way to potentially restore CSF folate levels in CFD patients with this impaired transport through the Folate Receptor alpha. This ‘rescue’ transport mechanisms of folate into the brain presumably relies on pharmacological doses of folate producing high enough levels that folate can be taken up into the brain across the blood brain barrier through other transporters, such as the Reduced Folate Carrier. In this 2005 seminal work, CFD patients treated with high dose folinic achieved exhibited levels of 5-mTHF in the CSF that matched controls and for some individuals, improvements in symptoms were reported.
In this seminal study of 28 CFD patients measuring folate receptor autoantibodies (FRAAs), the researchers noted that 4 of the 28 patients who had very high titers also had met the diagnostic criteria for autism and 2 of the 4 younger patients (I.e., those who presumably had less cumulative exposure to FRAAs) reportedly improved upon treatment with folic acid or folinic acid. This finding led to a hunt for whether FRAAs were prevalent in common ASD (without complication by an abject CFD diagnosis), whether a cerebral folate deficiency might be involved in the etiology of more general ASD, whether treatment with high dose folates to correct low brain folate levels might prove therapeutic for ASD symptoms and what the trigger for the FRAAs might be.
The research in this space has been pretty limited, not due to the quality of the researchers but rather the funding for this topic. If there’s one conclusion you walk away from this post with, I hope it’s that the topic of folate and autism is far from any conclusions but is deserving of more research funding. To date, cohorts examining FRAAs in cases and controls have been pretty small, but converge on relative 70% of individuals with ASD exhibiting at least one type of FRAA (there are both blocking and binding subtypes). This 70% is pretty high relative to the 15% estimated in non-sibling controls; however, the rates of FRAAs in parents and neurotypical siblings are 45% and 61%, suggesting a strong genetic component to forming them and their presence alone not being sufficient for an ASD diagnosis (just like our NTD story: variable risk penetrance that complicates who will manifest clinical symptoms and benefit from treatment).
Positivity for FRAAs is just one piece of the pie - the concentration of these autoantibodies (different thresholds have been proposed for blocking and binding types), as well as the time periods of exposure are likely to matter for how severe the phenotype is and how well individuals respond to treatment. At present, existing treatment trials are generally supportive of a response to supplementation with high dose, pharmacological preparations of folinic acid (there are about 4 randomized controlled trials so far in the general childhood ASD population) — but the field lacks a definitive blockbuster RCT. The available literature has largely been conducted by the same research group, enrolled <50 patients total in each trial, has varied in their assessment of changes in autism behaviors, used different doses of folinic acid, and have, at times, employed some subpar statistical practices (relying on post-hoc subgroup comparisons, as well as comparing within group changes, not changes relative to placebo). These limitations are all hallmark signs of an early and maybe promising hypothesis in need of really rigorously testing.
The good news is that there are some ongoing trials of folinic acid in autism but we need a whole bunch more. This variant of the folate-autism hypothesis is going to need multiple trials that measure outcomes in a similar manner, measure FRAAs, use similar doses of folinic acid, and employ other similar co-interventions. Several trials exist in the literature that employ folinic acid alongside a cocktail of other nutrients like methylcobalamin and employ a dairy-free diet as a background recommendation. Dairy is thought to be a major reason for the existence of FRAAs, because dairy contains a soluble ortholog of the folate receptor alpha that when consumed, causes some individuals to make antibodies against it that recognize not only this bovine isoform but also the human folate receptor alpha. This data for dairy as the cause of FRAAs comes from a small trial in patients with CFD and whether a dairy-free diet as an adjunctive therapy will prove beneficial for individuals with ASD and high FRAA titers remains to be seen.
For anyone excited about this hypothesis, which I am cautiously so, I think it’s important to push back on anyone who makes it sound solved - this is a hypothesis in need of testing and more clinical research to figure out what percentage of individuals with ASD might present with high FRAAs, low cerebral folate status and obtain benefits from high dose folate supplementation. Whether this is a small, unique subset of patients with an ASD diagnosis or a much more relevant to a larger percentage of individuals diagnosed with ASD is going to require much larger cohort studies and trials.
I would encourage any individuals with ASD or their caregivers interested in leucovorin/5-formylTHF to discuss this issue with their prescribing provider. There are going to be folks online who will hawk 5-formylTHF/folinic acid supplements but these have the usual quality issue of supplements as well as an issue of dose - current treatment protocols use doses an order of magnitude higher than supplements, up to 50mg, which would be a pricey option for potentially low quality supplements. Quality for 5-formylTHF is key given that there are different isomers, with only the L-isomer being biologically active. The pharmaceutical version, leucovorin, is a known 1:1 mix of d,l isomers and has been used for decades to improve folate status with a high dose. The supplements that are on the market often have limited information on quality and are not held to any meaningful standards like pharmaceutical preparations are, in addition to their being quite pricey when considering the need to meet the doses used in existing trials.
If there are clinicians out there, I do hope you’ll encourage patients to enroll in clinical trials if this is an option, and if not, write up a case study of the effects you observe (positive or negative). Patient-centered discussions around this topic will be key given the weak evidence, and in the event caregivers or individuals with ASD wish to trial leucovorin, I would encourage providers to gather n=1 data and write up case studies/series to objectively describe their experiences. I’ve already heard from patients in my DMs that some folks already tried it and had a range of experiences from no effect, to modest and dramatic. We want this sort of literature in the public knowledge sphere, especially at a time when there is political pressure to identify causes and ‘cures’ for autism.
A few final notes:
I made a post about this topic with Jessica Knurick PhD RD over on instgram and the responses from clinicians (naturopaths, ‘integrative’ practitioners) have been a bit all over the place but many are purporting that various supplements and diets work, with important effects of specific genotypes and FRAA testing. What is emerging amongst claims is essentially a quite complicated diet-x-supplemental form-x-genotype-x-FRAA interaction that no studies to date have assessed, or have been adequately powered to even look at. I appreciate clinical perspectives for what they are, but these are anecdotes from a subset of clinicians who are quite enthusiastic about theory over evidence-based practice. We should be clear that there is, at present, not evidence to support ordering MTHFR or other genotype testing or promoting heavily restrictive folic acid avoidance diets, and the evidence for FRAA testing and folinic acid treatment is quite limited. These hypotheses about optimal diagnosis and treatment approaches should be explored ideally in well-powered cohorts and trials. Of course, there will be patients who want to pursue treatments that are not currently recommended at-large and I am a big fan of patient-centered and led decision making, especially when the evidence is as limited as it is here — but a truly informed, patient-led decision making requires presenting the reality of where the data is at, minimizing your practitioner enthusiasm for a theory and focusing on rigorous, objective measures of the certainty of the evidence-base - overly enthusiastic clinicians who are more interested in treating based on hypothesis and pushing a brand of ‘integrative’ are impairing the ability of patients to make judgements about the (potentially pricey and burdensome) treatments/testing that are being pushed.
If the FRAA topic excites you, I understand! But id point you to the topic of FRAAs and neural tube effects as reason to stay measured and not expect this to be some magic bullet. An early small cohort reported FRAAs being elevated in pregnancies affected by NTDs, but subsequent cohorts have been mixed, with data from an Irish trial failing to find a link but a California cohort finding a modest relationship.
run, don’t walk, from anyone who tries to give diet or supplement advice based on the FRAA folate-autism story right now. I’ve already seen folks jumping to say this is another reason to avoid folic acid (there’s no good evidence for this) and others recommending 5-methylTHF supplements (which may be ok but have less data so far than leucovorin and again, are not sold at the doses being studied in trials to correct CSF folate levels). Folks are out to make money off the folate wars, good intentions or not, and this will be yet another opportunity. Given the uncertainty, all care not in a clinical should be patient-centered after genuine informed consent ,which includes being honest about the uncertainty in the evidence at the moment.
If patients are choosing to trial dairy free diets, it’s important to due this alongside a registered dietitian who can ensure that the diet remains adequate - dairy is often a major source of many essential nutrients for the pediatric population and this is often the case in individuals with ASD, so identifying nutritionally adequate replacements is key.
There is already a lot of potential for mommy-shaming and blaming emerging from the current focus on tylenol and folate during pregnancy. Shaming and blaming doesn’t help anyone, and the available data at the moment supports following current dietary guidelines. There is no evidence at present to support parents feeling guilty for using folic acid supplements as generally recommended during pregnancy with respect to ASD risk, and the market out there that makes parents feel guilty for this are usually trying to sell non-evidence-based genetic tests and supplement regimens, as well as restrictive diets to avoid folic acid. I’d also note that the FRAA literature has not really been explored during pregnancy yet with respect to offspring ASD risk, so any suggestions about FRAA testing and supplementation protocols are speculative at present.
I’ve seen quite a bit of misinformation going around from folks conflating ASD prevention and treatment, and folate deficiency with Cerebral folate deficiency. This has led to a whole bunch of information that isn’t really correct, like the below post stating that homocysteine levels should guide folinic acid treatment. If you recall, folinic acid is being used in ASD with FRAAs and a cerebral folate deficiency, a condition where serum folate status measures like folate levels and homocysteine look normal! Normal serum folate status markers shouldn’t be guiding folinic acid treatment - if anything, FRAA tests should, ideally in the context of a clinical trial.
Thank you so much for this detailed explanation that is easily understandable. It’s a huge help!