Consequences of folate deficiency – treated by immunomodulators (Infliximab, IVIG, Propes and Inflamafertin) and the relevance of mutations in MTHFR, MTR, and MTRR genes in identifying those at risk. Plus the effect of rTMS and tDCS on milder autism

 

Today’s post returns to folate deficiency, but before that a quick mention of magnetic/electrical brain stimulation therapies for autism without impaired cognition.

I encountered a new term IC-ASD. It stands for intellectually capable autism spectrum disorder. Most people with autism these days seem to have IC-ASD. Some struggle and some do not.

 

The effects of rTMS and tDCS on repetitive/stereotypical behaviors,cognitive/executive functions in intellectually capable children and young adults with autism spectrum disorder: A systematic review and meta-analysis of randomized controlled trials

 

Objective

This study aims to evaluate the efficacy of repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS) on repetitive/stereotypical behaviors and cognitive/executive functions in children and young adults with intellectually capable autism spectrum disorder (IC-ASD).

Methods

Literature searches across PubMed, Web of Science, Cochrane Library, Embase, and Scopus were performed to identify randomized controlled trials (RCTs) evaluating the efficacy of rTMS and tDCS in children and young adults with IC-ASD. The search encompassed articles published up to April 25, 2025. The standardized mean difference (SMD) with 95 % confidence intervals (CI) was calculated and pooled. Sensitivity and subgroup analyses were conducted to assess potential sources of heterogeneity and refine the robustness of the findings.

Results

This meta-analysis included 18 RCTs involving 813 participants. Compared with sham interventions, tDCS demonstrated significant improvements in social communication, repetitive and stereotypical behaviors, cognitive and executive functions among individuals with IC-ASD (e.g., Social Responsiveness Scale: SMD = –0.48; 95 % CI: –0.75 to –0.22; p < 0.01). Similarly, rTMS improved social communication, repetitive and abnormal behaviors (Social Responsiveness Scale: SMD = –0.21; 95 % CI: –0.42 to –0.00; p < 0.05; Repetitive Behavior Scale-Revised: SMD = –0.62; 95 % CI: –1.17 to –0.07; p = 0.04; Aberrant Behavior Checklist: SMD = –0.53; 95 % CI: –0.79 to –0.26; p < 0.01). No significant heterogeneity was observed across studies.

Conclusion

tDCS and rTMS may enhance cognitive and executive functions and reduce repetitive behaviors in children and young adults with IC-ASD. However, these findings require careful interpretation due to the limited high-quality studies and variability in treatment protocols. Future research should prioritize the development of standardized protocols to address inconsistencies in stimulation parameters (including frequency, intensity, and duration) and core outcome sets. Additionally, larger-scale, rigorously blinded multi-center RCTs are necessary to accurately evaluate the clinical efficacy and applicability of these neuromodulation techniques in these populations.

 

rTMS and tDCS look like interesting non-pharmaceutical options for those with milder types of autism. How well they work in those with lower cognitive function is not addressed.

 

Back to Folate Deficiency

Stephen recently highlighted a Chinese study that looked at the relevance of mutations in the genes MTHFR, MTR, and MTRR to try and identify those most at risk of folate deficiency.

I also highlight research into treating some of the downstream consequences that occur when folate metabolism is impaired. The lack of folate disrupts the immune system causing anomalies such as low NK cells, low NKT cells, high TNF-alpha.

Immunodeficiency (Low NK and NKT cells): The deficiency in these crucial innate immune cells means the body's ability to fight off infections (particularly opportunistic ones) and perform immune surveillance (e.g., against abnormal cells) is compromised. This immunosuppression is a direct consequence of the impaired cell proliferation due to the folate cycle defect.

Systemic Inflammation (High TNF-alpha): Despite the low numbers of certain immune cells, there can be an overproduction of pro-inflammatory cytokines like TNF-alpha. This leads to chronic systemic inflammation. This phenomenon is often referred to as hypercytokinemia.

Beyond TNF-alpha, you might expect a possible overproduction of:

• Interleukin-1 beta (IL-1β): This is a potent pro-inflammatory cytokine involved in various immune responses and neuroinflammation.
• Interleukin-6 (IL-6): Another major pro-inflammatory cytokine that plays a role in systemic inflammation and can affect brain development and function.
• Interferon-gamma (IFN-γ): This is a key cytokine in Th1 immune responses and is also pro-inflammatory.

 

The recent Chinese study concludes that high-dose folinic acid appears to be a promising intervention for children with autism. Its efficacy is notably associated with specific folate metabolism gene polymorphisms. The researchers suggest that high-dose folinic acid may help to improve neurodevelopmental outcomes by alleviating the folate metabolism abnormalities caused by single or combined mutations in these genes.

This research indicates that providing a metabolically active form of folate (folinic acid, calcium folinate, leucovorin etc) can be a direct approach to address the underlying metabolic challenges in a subset of people with autism who have specific genetic predispositions related to folate metabolism. Children with MTHFR A1298C or MTRR A66G mutations showed greater improvements in various developmental domains compared to those with the standard versions.

The intervention group demonstrated significantly greater improvements in social reciprocity compared to the control group.

No significant adverse effects were observed during the intervention period.

 

How does this fit in with US research into brain folate deficiency in autism

US researchers consider an autoimmune mechanism where the body produces antibodies that specifically target the Folate Receptor Alpha (FRα). FRα is a crucial protein responsible for transporting folate across the blood-brain barrier (and into other cells).

When these antibodies bind to FRα, they block or interfere with the normal transport of folate into the cells, particularly into the brain. This results in Cerebral Folate Deficiency (CFD), where folate levels in the cerebrospinal fluid are low, despite potentially normal folate levels in the blood.

US research indicates that FRAAs are prevalent in a significant percentage of children with ASD (up to 70% in some studies) and are associated with specific physiological and behavioral characteristics.

Treatment with folinic acid/ leucovorin has been shown to be effective in many children with autism who are positive for FRAAs, improving symptoms like communication, irritability, and stereotypical behaviors. It is believed that high doses of folinic acid can overcome the transport blockade caused by the antibodies

The US and Chinese research avenues complement each other by identifying different, but potentially converging, pathways that lead to folate dysfunction in autism, both of which demonstrate the therapeutic potential of folinic acid.

Here is the Chinese paper: 

Safety and Efficacy of High-Dose Folinic Acid in Children with Autism: The Impact of Folate Metabolism Gene Polymorphisms

Background/Objectives: Research on the safety and efficacy of high-dose folinic acid in Chinese children with autism spectrum disorder (ASD) is limited, and the impact of folate metabolism gene polymorphisms on its efficacy remains unclear. This trial aimed to evaluate the safety and efficacy of high-dose folinic acid intervention in Chinese children with ASD and explore the association between folate metabolism gene polymorphisms and efficacy. Methods: A 12-week randomized clinical trial was conducted, including 80 eligible children with ASD, randomly assigned to an intervention group (n = 50) or a control group (n = 30). The intervention group was administered folinic acid (2 mg/kg/day, max 50 mg/day) in two divided doses. Efficacy was measured using the Psycho-Educational Profile, Third Edition (PEP-3) at baseline and 12 weeks by two trained professionals blind to the group assignments. Methylenetetrahydrofolate reductase (MTHFR C677T, MTHFR A1298C), methionine synthase (MTR A2756G), and methionine synthase reductase (MTRR A66G) were genotyped by the gold standard methods in the intervention group. Results: 49 participants in the intervention group and 27 in the control group completed this trial. Both groups showed improvements from baseline to 12 weeks across most outcome measures. The intervention group demonstrated significantly greater improvements in social reciprocity compared to the control group. Children with MTHFR A1298C or MTRR A66G mutations demonstrated greater improvements in various developmental domains than wild type. Folinic acid may be more effective in certain genotype combinations, such as MTHFR C677T and A1298C. No significant adverse effects were observed during the intervention. Conclusions: High-dose folinic acid may be a promising intervention for children with ASD, and its efficacy is associated with folate metabolism gene polymorphisms. High-dose folinic acid intervention may promote better neurodevelopmental outcomes by alleviating folate metabolism abnormalities caused by single or combined mutations in folate metabolism genes.

 

Treating the downstream consequences of low brain folate

Today’s next papers highlight Infliximab, IVIG, Propes, and Inflamafertin as immunomodulatory therapies that target the downstream consequences of folate deficiency; they do not address or improve the underlying lack of folate.

Folate Deficiency in the Brain: This means there is an inherent problem in the body's ability to process or utilize folate, even if dietary intake is sufficient. It is often due to mutations in genes encoding enzymes of the folate cycle (like MTHFR) or transporters. This leads to issues with DNA synthesis, cell proliferation, and methylation, impacting various systems, including the immune system.

 

Infliximab

Infliximab is a TNF-alpha inhibitor. It blocks the activity of TNF-alpha, a key pro-inflammatory cytokine.

It does not put more folate into the system or fix how folate is metabolized. It is like putting out a fire (inflammation) that was started because of a broken electrical wire (folate deficiency's impact on immunity).

 

IVIG (Intravenous Immunoglobulin)

IVIG is a broad-acting immunomodulatory therapy composed of pooled antibodies from thousands of healthy donors. Its mechanisms are complex and include neutralizing autoantibodies, blocking Fc receptors, modulating cytokine production, affecting T and B cell function, and influencing complement activation.

IVIG aims to rebalance a dysregulated immune system, reduce inflammation, and sometimes provide passive immunity. It is like resetting an overactive or misdirected immune alarm system. The effect may not last.

 

Propes

Propes contains alpha- and beta-defensins and has a "pronounced immunoactivating and lymphoproliferative effect." It directly stimulates the growth and activity of immune cells like NK and NKT cells. It directly addresses the numbers and activity of NK and NKT cells that are deficient due to the folate cycle problem. It makes the existing cells (or promotes the creation of new ones) work better, despite the underlying folate issue.

 

Inflamafertin

This drug, containing alarmines and adrenomedulin of placental origin, has "pronounced anti-inflammatory and immunomodulatory effects mediated by the induction of interleukin 10 synthesis." Its role is to temper the immune activation  and ensure a more balanced, anti-inflammatory environment.

 

In summary

These therapies are all symptomatic or compensatory treatments for the consequences of genetic folate deficiency on the immune system and the body. They address the resulting immunodeficiency, inflammation, and associated clinical symptoms (like behavioral issues or opportunistic infections).

 

They do not:

• Add more folate to the body (like folic acid or L-methylfolate supplementation would).
• Correct the genetic defect that causes the folate cycle deficiency.
• Improve the body's intrinsic ability to metabolize folate.

Genetic deficiency in the folate cycle disrupts fundamental cellular processes required for the normal development, proliferation, and function of NK and NKT cells, leading to their deficiency in affected children. This deficiency, in turn, contributes to the complex immune dysregulation often seen in autism.

 

Key Findings on NK Cells:

• Initial Deficiency: A significant number of children in the study group (53 patients) had an initial deficiency of NK cells.
• Response to Immunotherapy:
• During the 3-month course of Propes and Inflamafertin, the average number of NK cells in the blood almost doubled.
• NK cell counts reached the lower limit of normal in 74% (39 out of 53) of the patients with a deficiency.
• There was a strong statistical link between the immunotherapy and NK cell normalization.
• Sustainability: A notable finding was that the NK cell numbers returned to almost their initial level within 2 months after the immunotherapy was stopped. This suggests that the effect on NK cells might be temporary and dependent on continuous treatment.

 

Key Findings on NKT Cells:

• Initial Deficiency: A larger proportion of children in the study group (87 patients) had an initial deficiency of NKT cells.
• Response to Immunotherapy:
• The average number of NKT cells in the blood increased by half during the 3-month immunotherapy course.
• NKT cell counts were normalized in 89% (78 out of 87) of the patients with a deficiency.
• There was an even stronger statistical link between the immunotherapy and NKT cell normalization compared to NK cells.
• Sustainability: Importantly, the NKT cell numbers continued to grow for an additional 2 months after the discontinuation of the immunotropic drugs. This suggests a more sustained and potentially longer-lasting effect on NKT cells.

Overall Conclusions from the Study:

• Combination immunotherapy with Propes and Inflamafertin is presented as an effective treatment strategy for the immunodeficiency (specifically NK and NKT cell deficiency) found in children with ASD linked to genetic folate deficiency.
• Both biological drugs were able to normalize the reduced numbers of NK and NKT cells during the 3-month treatment period.
• The study highlights that the effect on NKT cells was more frequent, stronger, and more lasting compared to the effect on NK lymphocytes.

 

The research papers:

EFFICACY OF INFLIXIMAB IN AUTISM SPECTRUM DISORDERS IN CHILDREN ASSOCIATED WITH GENETIC DEFICIENCY OF THE FOLATE CYCLE

 The notion of systemic inflammation in autism spectrum disorders in children has been established. A recent meta-analysis of randomized controlled trials published in 2019, which included a systematic review of 25 case-control studies, suggests an association between genetic deficiency of the folate cycle and autism spectrum disorders in children [18]. This evidence is consistent with an earlier meta-analysis of randomized controlled trials from 2013, which included data from 8 studies [17]. The encephalopathy that develops in children with genetic deficiency of the folate cycle and manifests as autism spectrum disorders is associated with oxidative stress. The reason for the latter can be seen in the suppression of the immune system with the development of a special form of immunodeficiency, which is based on the deficiency of natural killers, natural killer T lymphocytes and CD8 +  cytotoxic T cells [11]. Immunodeficiency mediates all three known mechanisms of brain damage in children with genetic deficiency of the folate cycle, namely the development of opportunistic infections [2, 15], autoimmune reactions against neuronal antigens [3, 6] and manifestations of systemic inflammation, which is based on the phenomenon of hypercytokinemia [13, 20]. Children with autism spectrum disorders have been shown to have overproduction of several proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta, and interleukin-6

In SG, there was a pronounced positive dynamics in the direction of hyperactivity, hyperexcitability and stereotyped behavior, but no significant effect was noted on the stability of eye contact and the development of expressive-receptive language, while in CG some positive changes were achieved specifically in terms of expressive language and the level of eye contact, which indicates different points of action of infliximab and specialized educational programs (Table 11.1). The psychotropic effect obtained with infliximab differs from that of intravenous immunoglobulin, which has also demonstrated clinical efficacy in ASD associated with GDFC [10, 12]. The changes induced by infliximab are more pronounced and develop in a shorter time frame, but they are significantly narrower in terms of the spectrum of positive psychotropic effects compared to high-dose immunoglobulin therapy, which has a total modifying effect on the psyche of such children.

Materials and methods. This prospective controlled single-center non-randomized clinical study included 225 children diagnosed with autism spectrum disorders associated with genetic deficiency of the folate cycle. The diagnosis of autism spectrum disorders was made by psychiatrists from regional hospitals or specialized departments according to DSM–IV–TR (Diagnostic and Statistical Manual of mental disorders) and ICD–10 criteria. Children were recruited into the study group (SG) in 2019–2020. These were patients from different regions of Ukraine aged 2 to 9 years, in whom elevated serum TNF-alpha concentrations were observed. As is known, the phenotype of genetic deficiency of the folate cycle includes 5 main syndromes: autism spectrum disorders, intestinal syndrome (persistent enteritis/colitis) [7], PANDAS [4, 9], epileptic syndrome [5] and signs of pyramidal tract damage.

 

Conclusions. Infliximab leads to significant improvements in hyperactivity and hyperexcitability, as well as stereotypic behavior in children with autism spectrum disorders associated with genetic deficiency of the folate cycle. Responders to immunotherapy are 76 % of patients with this pathology, which is twice as high as with standard therapy. However, there is no effect of infliximab on such manifestations of autism as the level of eye contact and language development. Psychotropic effects of infliximab immunotherapy are closely related to the normalization of previously elevated serum TNF-alpha concentrations and are probably due to the elimination of the pathological activating effect of this pro-inflammatory cytokine on CNS neurons. In parallel, there is an improvement in other clinical syndromes of genetic deficiency of the folate cycle in children with autism spectrum disorders – intestinal pathology, epileptic syndrome, and PANDAS, in the pathogenesis of which, as is known, TNF-alpha and the systemic and intracerebral inflammation induced by this cytokine are involved. However, under the influence of immunotherapy, there is no change in the dynamics of motor deficit in children with symptoms of pyramidal tract damage. Further clinical studies in this direction with a larger number of participants and randomization are necessary to obtain more convincing data.

Efficacy of combined immunotherapy with Propes and Inflamafertin in selective deficiency of NK and NKT cells in children with autism spectrum disorders associated with genetic deficiency of the folate cycle

 Objectives. The results of previous small clinical trials indicate the potential benefit of combination immunotherapy with Propes and Inflamafertin to compensate for NK and NKT cell deficiency due to genetic deficiency of the folate cycle in children with autism spectrum disorders. The purpose of the research was to study the effectiveness of combined immunotherapy with Propes and Inflamafertin in NK and NKT cell deficiency in children with autism spectrum disorders associated with genetic deficiency of the folate cycle. Material and methods. This single-center, prospective, controlled, nonrandomized clinical trial included 96 children aged 2 to 10 years with autism spectrum disorders associated with a genetic folate deficiency (study group, SG). Children of SG received Propes at a dose of 2 ml IM every other day for 3 consecutive months (45 injections), and Inflamafertin at a dose of 2 ml IM every other day for 3 months in a row, alternating with Propes (45 injections). The control group (CG) consisted of 32 children of similar age and gender distribution who suffered from autism spectrum disorders associated with genetic deficiency of the folate cycle, but who did not receive immunotherapy. Outcomes. The number of NK cells reached the lower limit of normal in 39 out of 53 patients (74% of cases), with the resulting deficiency of these lymphocytes, and the average number of NK cells in the blood in SG almost doubling during the 3-month course of immunotherapy (р ˂ 0.05; Z ˂ Z0.05). However, it returned to almost initial level in the 2 months following the discontinuation of immunotherapeutic agents (р˃0.05; Z˃Z0.05). The number of NKT cells was normalized in 78 out of 87 patients (89% of cases) with an initial deficiency of these cells, and the average number of NKT cells in the blood in the DG increased during the course of immunotherapy by half (р ˂ 0.05; Z ˂ Z0.05) and continued to grow for the next 2 months after the discontinuation of immunotropic drugs (р ˂ 0.05; Z ˂ Z0.05). There was a link between immunotherapy and normalization of NK - (χ2 = 18.016; OR = 13.929; 95%CI = 3.498-55.468) and NKT-cells (χ2 = 60.65; OR = 46.800; 95%CI = 14.415-151.937) in the blood with a strong association between these processes (criterion φ = 0.504 and 0.715 respectively; С = 0.450 and 0.581 respectively). Conclusions. Combination immunotherapy with Propes and Inflamafertin is an effective strategy for the treatment of immunodeficiency caused by genetic deficiency of the folate cycle in children with autism spectrum disorders.

 

The results obtained in this controlled non-randomized clinical trial indicate that combination immunotherapy with Propes and Inflamafertin is an effective treatment strategy for immunodeficiency caused by genetic folate deficiency in children with autism spectrum disorders. These biological immunotropic drugs are able to normalize the previously reduced number of NK and NKT cells in the blood in this category of patients during a 3-month course of immunotherapy, with a more frequent, stronger and more lasting effect on NKT cells compared to NK lymphocytes.

  

Conclusion

Folinic acid supplementation is an effective therapy for many people with autism. There are many anomalies that appear, for example those people who test positive for the folate transporter antibodies but a lumbar punction then finds normal levels of folate in the brain.  Many people report agitation or aggression when children take calcium folinate at high doses, but this does not seem to get noted in clinical trials. Nonetheless it looks like everyone with autism should at least make a trial.

Note that you should always add a vitamin B12 supplement when giving high dose calcium folinate. This is because more B12 will be required by the biological processes ongoing in the brain and deficiency will cause side effects.

Many people who respond well to calcium folinate end up needing some kind of immunotherapy on top. IVIG is extremely expensive and quite a bother if you need to take it forever. Some of the therapies from the two papers today also involve a very large number of injections, so are not really practical.  The less intrusive immunotherapies look more practical but are not cheap.

I think that rTMS and tDCS will be attractive to those seeking non-pharmaceutical options that have a scientific basis. The same applies to low level laser therapy, also known as photobiomodulation therapy.

Bumetanide - Biomarkers from Shanghai for Autism responders

 

“three cytokine levels, namely the IFN-γ, MIG and IFN-α2  … These cytokine levels at the baseline could improve the prediction of the bumetanide responders”

“… cytokines had a potential to construct a blood signature for predicting and monitoring the bumetanide treatment in young children with ASD.”

“a significant part of the clinical heterogeneity in the treatment effect of bumetanide for ASD is associated with the differences in the immune system of patients”

 

Autism is a very heterogeneous family of conditions and this is a big part of the reason why all clinical trials to date have failed.  Ideally, there would be a diagnostic test to identify which person will respond to which therapy.  Then you can have a successful clinical trial, because you are only including people likely to respond.

Researchers from China have just published their results that suggest that a blood test measuring three inflammatory markers can predict who will respond to bumetanide.  This is good news and where it is coming from is also very notable.

Autism research has been very fragmented, some of it is very sophisticated and insightful but much is very amateur and some is quite trivial.  There is usually a real lack of common sense among these people and no sense of urgency whatsoever.

China is a very organised country; plans are made and then they are implemented.  Forget political correctness.

This kind of approach is what is required to move along with autism treatment.

In addition, there is also another new study from China, this time on the microbiota in autism that compared those with and without GI problems (it found it is equally disturbed in both groups). Hopefully, that Chinese group will do the next common sense step and compare the microbiota of autistic people with and without restrictive diets. To what extent to people give themselves a microbiota problem through poor diet.

 

Disentangling the relationship of gut microbiota, functional gastrointestinal disorders and autism: a case–control study on prepubertal Chinese boys

The altered gastrointestinal microbiota composition in ASD appeared to be independent of comorbid functional gastrointestinal disorder

 

The bumetanide researchers are from Fudan University in Shanghai, one of the 3 ultra-selective Chinese Universities alongside Tsinghua University and Peking University in Beijing.

The paper, not surprisingly, may look complicated, but there are a great deal of interesting things in it.

In their words:-

An immuno-behavioural covariation was identified between symptom improvements in the Childhood Autism Rating Scale (CARS) and the cytokine changes among interferon (IFN)-γ, monokine induced by gamma interferon and IFN-α2. Using this covariation, three groups with distinct response patterns to bumetanide were detected

The three groups were: best responders, least responders and medium responders.

It should be noted that the dosage used in their trials was 0.5mg of bumetanide twice a day.

Chinese children tend to be smaller than Western children and this might help explain why the results were more positive than in Servier’s failed phase 3 clinical trial in Europe. I also imagine the Chinese children were more severely autistic than the European group.

The dosage used is selected to minimize the diuresis rather than to maximize the impact on the autism. This is understandable, but I think it is a mistake.

 

The immuno-behavioural covariation associated with the treatment response to bumetanide in young children with autism spectrum disorder 

Bumetanide, a drug being studied in autism spectrum disorder (ASD) may act to restore gamma-aminobutyric acid (GABA) function, which may be modulated by the immune system. However, the interaction between bumetanide and the immune system remains unclear. Seventy-nine children with ASD were analysed from a longitudinal sample for a 3-month treatment of bumetanide. The covariation between symptom improvements and cytokine changes was calculated and validated by sparse canonical correlation analysis. Response patterns to bumetanide were revealed by clustering analysis. Five classifiers were used to test whether including the baseline information of cytokines could improve the prediction of the response patterns using an independent test sample. An immuno-behavioural covariation was identified between symptom improvements in the Childhood Autism Rating Scale (CARS) and the cytokine changes among interferon (IFN)-γ, monokine induced by gamma interferon and IFN-α2. Using this covariation, three groups with distinct response patterns to bumetanide were detected, including the best (21.5%, n = 17; Hedge’s g of improvement in CARS = 2.16), the least (22.8%, n = 18; g = 1.02) and the medium (55.7%, n = 44; g = 1.42) responding groups. Including the cytokine levels significantly improved the prediction of the best responding group before treatment (the best area under the curve, AUC = 0.832) compared with the model without the cytokine levels (95% confidence interval of the improvement in AUC was [0.287, 0.319]). Cytokine measurements can help in identifying possible responders to bumetanide in ASD children, suggesting that immune responses may interact with the mechanism of action of bumetanide to enhance the GABA function in ASD.

 

The use of bumetanide as a potential drug to improve symptoms in ASD is based on a hypothesised pathoetiology of ASD, namely the delayed developmental switch of the gamma-aminobutyric acid (GABA) functioning from excitatory to inhibitory [10,11,12]. In the valproate and fragile X rodent models of autism, this GABA-switch can be facilitated by the reduction of intracellular chloride concentration, which is mediated by a sequential expression of the main chloride transporters, such as the potassium (K)-Cl co-transporters 2 (KCC2) and the importer Na-K-Cl cotransporter 1 (NKCC1) [12]. Therefore, bumetanide as an NKCC1 inhibitor has been tested for its ability to restore GABA function in ASD [5,6,7, 13, 14]. However, these transporters can also be influenced by other molecules, such as cytokines, which are a number of small cell-signalling proteins closely interacting with each other to modulate the immune reactions. The cytokines have been implicated not only in brain development [15], but also in GABAergic transmission [16,17,18]. It has been reported that the interferon (IFN)-γ can decrease the levels of NKCC1 and the α-subunit of Na⁺-K⁺-ATPase, contributing to the restore of inhibitory GABA function [16]. In mice subjected to maternal deprivation, the interleukin (IL)-1 has also been found to reduce the expression of KCC2, delaying the developmental switch of the GABA function and thereby possibly contributing to the pathophysiology of developmental disorders such as ASD [17, 18]. Therefore, a question naturally arises that whether the treatment effect of bumetanide for ASD can be affected by the immune responses in the patients.

Indeed, compared with healthy controls, changes of the cytokine levels have already been reported in patients with ASD [19,20,21,22]. Recent meta-analyses showed that the levels of anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (Ra) were decreased [20], while proinflammatory cytokines IL-1β, IL-6 and anti-inflammatory cytokines IL-4, IL-13 were elevated in blood of patients with ASD [21]. The levels of IFN-γ, IL-6, tumour necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-8 were observed to be elevated [22] in postmortem brain tissues of ASD patients, and increased level of IFN-γ, monocyte chemotactic protein (MCP)-1, IL-8, leukaemia inhibitory factor (LIF) and interferon-gamma inducible protein (IP)-10 were found in another study [23]. These widely spread changes suggest that the cytokine signalling in ASD may be better characterised by multivariate patterns of cytokines. In literatures, many associations had been reported between the levels of cytokines (e.g., MCP-1, IL-1β, IL-4, IL-6, etc.) and both core symptoms and adaptive functions in children with ASD [24,25,26]. Therefore, it has been suggested that cytokines may be used as biomarkers to identify different subsets within ASD. In each of these subsets the patients with ASD may share a commonly immune-related pathoetiology and therefore may have similar profiles of response to treatment [27].

Based on these previous findings, we analysed data acquired through the Shanghai Xinhua ASD registry, China, that began in 2016 to test the hypothesis that the immune activity of patients might help to identify the best responders to bumetanide in ASD.

 

Between May 1st, 2018, to April 30th, 2019, a total of 90 ASD children, aged 3–10 years old, under a 3-month stable treatment of bumetanide without behavioural interventions and any concomitant psychoactive medications had both blood draws and behavioural assessments. Among these patients, 11 of them were further excluded due to the lack of the follow-up data at month 3. A group of 37 children, under 3-month stable treatment of placebo without behavioural interventions and any concomitant psychoactive medications had both blood draws and behavioural assessments. Therefore, the current analysis used a subsample of 116 young children with ASD, whose blood samples were available both before and after the treatment. The blood samples were sent in three batches (Discovery Set: n = 37 on December 4, 2019; Validation Set: n = 42 on May 22, 2019; and Control Set: n = 37 on January 5, 2022) to measure the serum levels of 48 cytokines for the immune response (Table S1), and the clinical symptoms were assessed using CARS, ADOS and the Social Responsiveness Scale (SRS). 

In this study, we observed a significant improvement of clinical symptoms with bumetanide treatment in children with ASD, and such improvement was associated with a pattern of changes in three cytokine levels, namely the IFN-γ, MIG and IFN-α2 (r = 0.459 in the Discovery Set and r = 0.316 in the Validation Set). These cytokine levels at the baseline could improve the prediction of the bumetanide responders compared with using the behavioural assessments alone, and the best predictor achieved an AUC of 0.83 in the independent test data set (Table S8). The implications of these findings may be twofold: (1) a significant part of the clinical heterogeneity in the treatment effect of bumetanide for ASD is associated with the differences in the immune system of patients, and (2) the component score of cytokines had a potential to construct a blood signature for predicting and monitoring the bumetanide treatment in young children with ASD.

Following the protocols of previous studies [8], bumetanide treatment consisted of two 0.5 mg tablets per day for three months, given at 8:00 a.m. and 4:00 p.m. The tablet size is 8 mm diameter x 2 mm thickness, which is quite small. Each time, the patient took half of a tablet, which was not difficult for most of the patients. However, the careers were recommended to grind the half-tablet into powder and give the powder in water, if necessary. Possible side effects were closely monitored during the treatment. Blood parameters (serum potassium and uric acid) were monitored via laboratory tests (Table S2) and symptoms (thirst, diuresis, nausea, vomiting, diarrhoea, constipation, rash, palpitation, headache, dizziness, shortness of breath, and any other self-reported symptoms) were telephone interviewed (Table S3), and both of them were reported to the research team by telephone at 1 week and 1 month after the initiation of treatment and at the end of the treatment period. The cytokine levels of the children with gastrointestinal problems were compared with those without such problems (Table S4).

The supplemental table S4 shows that GI problems had no effect on cytokine levels.

Changes after the administration of bumetanide

Seventy-nine patients were treated with bumetanide for 3 months, and the CARS total score decreased after the treatment (effect size Cohen’s d = 1.26, t₇₈ = 11.21, p < 0.001). The treatment effect showed no difference between the Discovery Set and the Validation Set (ΔCARS_total: mean(±SD): 1.54 (±1.40) vs. 1.90 (±1.34)). Consistent to the previous studies of the low-dose bumetanide for ASD, the side effects were rarely reported (Tables S2 and S3). No significant difference in the cytokine levels between the children with and without the gastrointestinal problems at the baseline (Table S4). A number of cytokine levels were changed significantly after the treatment of bumetanide, but none of them was changed significantly after the treatment of placebo (Table S6). No significant pairwise association could be identified in the Discovery Set, the Validation Set and the Control Set among four groups of variables, including the baseline CARS total score, the baseline cytokine levels, the change of CARS total score, and the changes of cytokine levels (Fig. S2).

 

In this study, we observed a significant improvement of clinical symptoms with bumetanide treatment in children with ASD, and such improvement was associated with a pattern of changes in three cytokine levels, namely the IFN-γ, MIG and IFN-α2 (r = 0.459 in the Discovery Set and r = 0.316 in the Validation Set). These cytokine levels at the baseline could improve the prediction of the bumetanide responders compared with using the behavioural assessments alone, and the best predictor achieved an AUC of 0.83 in the independent test data set (Table S8). The implications of these findings may be twofold: (1) a significant part of the clinical heterogeneity in the treatment effect of bumetanide for ASD is associated with the differences in the immune system of patients, and (2) the component score of cytokines had a potential to construct a blood signature for predicting and monitoring the bumetanide treatment in young children with ASD.

Accumulating evidences support that IFN-γ can inhibit chloride secretion [38] and down-regulate both the NKCC1 expression [16, 38] and the Na⁺-K⁺-ATPase expression [16], which had been implicated in the GABAergic dysfunction in ASD [10, 39].

 

The cytokine-symptom association was identified in the changes after the treatment of bumetanide but not before the treatment, suggesting that bumetanide might interact with the cytokines and the changes of which contributed to the treatment effect of bumetanide. Animal studies showed a rapid brain efflux of bumetanide, but a number of clinical trials have shown a significant treatment effect for neuropsychiatric disorders, including ASD, epilepsy and depression [41, 42]. These findings may suggest the possible systemic effects of bumetanide as a neuromodulator for these neuropsychiatric disorders. Considering its molecular structure, bumetanide has been recently identified by an in vitro screen of small molecules that can act as an anti-proinflammatory drug via interleukin inhibition [43]. This anti-proinflammatory activity of bumetanide might alter the blood levels of cytokines outside the brain-blood-barrier (BBB).

Our findings may suggest that the identified canonical score of cytokines had a potential to construct a blood signature for predicting and monitoring the bumetanide treatment in young children with ASD. Accurately identifying patients who are likely to respond positively to bumetanide can facilitate the precision medicine for ASD. Our prediction model based on the cytokine levels before the treatment may provide a potentially new tool for the precision medicine of ASD. 

 

In summary, we identified an association between the changes of the cytokine levels and the improvements in symptoms after the bumetanide treatment in young children with ASD, and found that the treatment effect of bumetanide can be better characterised by an immuno-behavioural covariation. This finding may provide new clinically important evidence supporting the hypothesis that immune responses may interact with the mechanism of bumetanide to restore the GABAergic function in ASD. This finding may also have relevance for determining enriched samples of ASD children to participate in novel drug treatment studies of drugs with a similar mode of action to bumetanide, but with potentially greater efficacy and fewer side effects.

 

Conclusion

I think we can give the Shanghai researchers 10 out of 10 for their paper.

Monty, aged 18 with ASD, has been to Shanghai twice. It is a vast city, but well worth a visit. With the high speed train network it is now very easy to travel around China, quite different to when I visited as a teenager.

Hopefully the Chinese will continue in their pursuit of precision medicine for autism. They do not have much competition.

My perspective is a little different because I know that a bumetanide responder can cease to be a responder when affected by an inflammatory condition like allergy, which increases pro-inflammatory cytokines like IL-6. This suggests that some people with elevated cytokines are potential responders, you just have to use an anti-inflammatory therapy before you start bumetanide therapy. The inflammatory cytokines shift the balance between NKCC1 and KCC2 towards NKCC1 and so increasing intracellular chloride.  We also know that some people need a dose higher than 0.5mg twice a day to see a large benefit; I have been using 2mg once a day for several years.

The Chinese researchers have established biomarkers for who is likely now to respond to bumetanide. This certainly is a big step forward, if it can be replicated. This is not the same as identifying who could respond to bumetanide, if their current inflammatory condition was moderated. The levels of specific cytokines might indeed mark someone as both a current non-responder, but also as a potential future responder.

Autism is all about n=1, it is about the exceptions being more important than the average.

Unlike the Shanghai researchers, I do not really see Bumetanide as an anti-inflammatory therapy in my son’s Polypill, but I do have therapies included that are.

Understanding inflammation will be a key to treating autism using precision medicine.  That is less simple that it sounds. When it comes to preventing autism, inflammation in the mother is a key part of the equation. This also gets complicated, maternal antibodies damage the brain of the fetus, no genetic mutations were needed.