“We did genetic testing and it came back clear!” Well your VCF file probably says otherwise!

 

In the TikTok/Instagram world where many people live these days, everything is kept very simple. The real world is becoming the alternative reality.

Over the years, many people have sent me their genetic testing results. Nowadays we have moved on to whole genome sequencing (WGS), which generates vast amounts of data and is pretty much as good as it gets. Vast amounts of data brings with it the problem of how to filter through it and not miss something critical.

I have written many times that parents who have had genetic testing carried out on their child should ask for the full list of mutations, not just those on the lab’s list for autism. In the case of whole exome sequencing (WES), this could produce a list of 10 to 30 mutations, which might well include an ion channel, or a similar variant, that is actually relevant to autism.  

More and more parents are doing this or even repeating the WGS elsewhere and then getting a very different interpretation, with a likely causal gene identified.

 

“We did genetic testing and it came back clear!”

I am sorry to disappoint the TikTokers, but nobody’s WGS results can come back clear.

 

What is whole genome sequencing (WGS)?

Scientists have put together a reference genome, based on the results of many different real people. 

In WGS, blood or saliva is used to sequence the entire genome of the patient, and then it is automatically compared to the reference genome. Tens of thousands to millions of variants will be identified. They all end up in the VCF (Variant Call Format) file.

 

The VCF (Variant call format) file

The VCF file is generated by special software. It contains details about the genetic variants.

 

Annotated VCF file

More automation then looks up each of the detected variants to see if they are already listed in databases, as being known to cause problems. If the variant has not been listed in these databases, it will not be highlighted.

These days most labs will provide the annotated VCF file and it can be huge.

 

The role of the geneticist or ChatGPT

The geneticist is then supposed to take the annotated VCF file and filter the results based on the clinical condition of the patient.

Results are categorized as:

·        Pathogenic

·        Likely pathogenic

·        Variant of uncertain significance

·        Likely benign

·        Benign

In theory, the geneticist’s job is to translate the vast and complex information from genetic tests into actionable insights for diagnosis, treatment, and genetic counselling, ensuring both precision and empathy in patient care.

He/she should check whether the identified variant explains the patient's specific symptoms (e.g., developmental delay, speech impairment, intellectual disability).

If multiple variants are identified, consider whether they might interact.

Some individuals may have only one variant, while others may carry several genetic changes that contribute to their condition.

 

How come there are so many apparently sloppy geneticists?

Doctors like to deal with certainties and the geneticist has to avoid diagnosing a gene as causal, when there is a chance it actually is not. They also know that there are almost no genes related to autism that they can treat. There is no incentive a take a stab at causality, when they know they cannot offer any follow-on treatment. Contrast that with the parent's perspective looking for any clues.

Genetics is actually all about probabilities, much more than certainties. So, if you have 3 mutations that are each individually “survivable”, the combination might be causal.

Here is an example:

Imagine a scenario where:

• Mutation 1: Causes a minor disruption in protein folding (survivable).
• Mutation 2: Impairs a metabolic enzyme function (survivable).
• Mutation 3: Affects a signaling pathway that slightly reduces cellular repair efficiency (survivable).

Each of these mutations, on their own, may not lead to a disease. However, when combined, these disruptions might overwhelm cellular systems and cause a disease phenotype (for example, a neurodegenerative condition), as the cumulative effect of these disruptions could impair essential cellular functions beyond a survivable threshold.

  

Not enough people with autism submit WGS data

I would think you need to have the WGS data from at least a million people with an autism diagnosis, from mild to severe, to be pretty sure you have identified the majority of causal mutations. Many of these causal mutations will actually be combinations of a few different genes - it would get very complicated. 

The number of people with autism currently included in the reference databases so far is tiny.  It is biased towards those with profound single-gene autism.

We really need to know about polygenic autism, which accounts for the vast majority of cases. Those cases range from profound to trivial.

I think that if I were in charge, I would tell people that if they want an autism diagnosis, they need to provide a saliva sample. No sample, no diagnosis. The test can be anonymous, if people prefer, so there is nothing to fear. At least this would reduce the waiting time for diagnosis!

  

TikTok autism

On social media you get a lot of self-diagnosed autism, but you also get some Moms/Mums of kids with profound autism.

I saw one today who has 3 sons and 1 daughter, all with profound autism. She says it cannot be genetic, because the geneticist did not find a causal gene.  In theory she might be right. Perhaps there is an environmental explanation, maybe the babies were all exposed to the same toxic environment (food, water, air, a high voltage power cable over the house …)

More likely, the geneticist did not do his job. Or, the kids have mutations that have not yet been added to the list of causal genes. The list grows every day. If she used a good provider, like GeneDX, and she ticked a box on the form, then they may come back to her in 5 years’ time and tell her that one of her kids’ mutations is now recognized as causal. This happened to one reader of this blog.   

My tip would be to ask for the Annotated VCF file and get ChatGPT to analyse it for anything that might explain profound autism.

It looks like any computer-savvy middle-aged person can do this; no prior experience needed!

 

Conclusion

I was pleasantly surprised to hear from several readers recently who followed my suggestion to dig deeper into the results of their child’s genetic testing. They all found something valuable, hidden away. Perhaps you wouldn’t contact Peter if you didn’t find something of use!

To analyze genetic data effectively, you will need the paid version ChatGPT Plus (GPT-4). While it is powerful, it does have processing limits. For example, processing a large VCF file requires breaking it into smaller, manageable parts. Fortunately, ChatGPT can guide you through this process.

If you were doing this as a professional service, you would be better off using GPT-4 via API. This tool, created by the same company, is designed for heavy data processing rather than conversational interaction.

When it comes to genetic forms of autism with approved drug therapies, there seem to be only two: 

·        Tuberous Sclerosis Complex (TSC1 or TSC2 Mutation): Treated with Everolimus (Afinitor) in the US, Canada, EU, UK, and Australia. 

·        Rett Syndrome (MECP2 Mutation): Treated with Trofinetide (Daybue) in the US and Canada if you have the money.

This highlights why geneticists focus on diagnosis rather than treatment. 

Unofficially, the possibilities for treatment are extensive, limited only by creativity and emerging research. Here again, ChatGPT can do much of the work for you.

P.S.  TikTok, Instagram and Facebook can be fun, but for something factual, better stick to ChatGPT or similar services.

P.P.S. I do not have shares in OpenAI, who own ChatGPT 

Low-dose clonazepam for autism - SCN2A deficient, SCN1A deficient, BTBR polygenic autism and Maternal Immune Activation (MIA) all respond to the same cheap treatment

 

Restoring the excitation/inhibition balance in neurons is a good way to treat both epilepsy and autism. Professor Catterall performed the groundwork in one model of epilepsy and one model of autism more than a decade ago, using low-dose clonazepam.

At that point I did endeavour to translate that science from mouse to human, in part because Professor Catterall made clear he was not going to.

A number of readers of this blog, biased towards doctor parents, did use this therapy for several years.

In 2021 the Chinese showed low-dose clonazepam effective in the Maternal Immune Activation (MIA) model of autism.

Roll forward to 2025 and Chinese researchers looking into another single gene autism (SCN2A) have found the same therapy to be effective.  Fancy that !!

It is actually the cheapest therapy I ever investigated, costing a few dollars/euros/pounds a year.

At a tiny dose, clonazepam, which acts as a positive allosteric modulator of GABA receptors, enhances the activity of receptors containing α2 and α3 subunits and restores the excitation/inhibition balance.

The effective dose in us humans is about 0.0006 mg/kg per day. Due to its long half-life, you need to take the dose for 3 days before the level in the body rises to the therapeutic level. As suggested by Catterall, there is a narrow therapeutic window; too high a dose, or too low a dose, will not be effective.

I used tablets, but it is much easier to use the liquid version of clonazepam, since you need to produce a very dilute version and then measure the dose with a syringe. Some people used a compounding pharmacy to do the hard work.

Not everyone responds and I think not everyone finds their therapeutic dosage window. I think people may need to adjust the dosage over the years.

There are many posts in this blog that refer to this therapy.

 

https://www.epiphanyasd.com/search/label/Clonazepam

 

What are the effects in children?

The effects are improved cognition, ability to learn new skills and a reduction in broad symptoms of autism.

In kids already taking bumetanide, and are responsive to that therapy, there is an additional benefit. In our case the incremental effect was less than bumetanide, but welcome nonetheless.

Some people use it as an alternative to bumetanide, when diuresis is problematic.

The dose is so low, the usual risks of benzodiazepines are not present. It might be better described as a micro-dose.

 

 The science, for those who are interested:

 

The recent paper from 2025:

  

Restoration of excitation/inhibition balance enhances neuronal signal-to-noise ratio and rescues social deficits in autism-associated Scn2a-deficiency

Social behavior is critical for survival and adaptation, which is profoundly disrupted in autism spectrum disorders (ASD). Social withdrawal due to information overload was often described in ASD, and it was suspected that increased basal noise, i.e., excessive background neuronal activities in the brain could be a disease mechanism. However, experimental test of this hypothesis is limited. Loss-of-function mutations (deficiency) in SCN2A, which encodes the voltage-gated sodium channel Na_V1.2, have been revealed as a leading monogenic cause of profound ASD. Here, we revealed that Scn2a deficiency results in robust and multifaceted social impairments in mice. Scn2a-deficient neurons displayed an increased excitation-inhibition (E/I) ratio, contributing to elevated basal neuronal noise and diminished signal-to-noise ratio (SNR) during social interactions. Notably, the restoration of Scn2a expression in adulthood is able to rescue both SNR and social deficits. By balancing the E/I ratio and reducing basal neuronal firing, an FDA-approved GABA_A receptor-positive allosteric modulator improves sociability in Scn2a-deficient mice and normalizes neuronal activities in translationally relevant human brain organoids carrying autism-associated SCN2A nonsense mutation. Collectively, our findings revealed a critical role of the Na_V1.2 channel in the regulation of social behaviors, and identified molecular, cellular, and circuitry mechanisms underlying SCN2A-associated disorders.

HIGHLIGHTS

1.     Na_V1.2 deficiency leads to pronounced social deficits in mice.

2.     Na_V1.2 deficiency results in an overall enhanced E/I ratio, elevated basal neuronal activity, and impaired signal-to-noise ratio.

3.     Both the enhanced E/I ratio and impaired sociability are reversible through the restoration of Na_V1.2 expression in adulthood.

4.     Targeted restoration of Na_V1.2 in striatum-projecting neurons rescues social impairments.

5.     GABA transmission is reduced in both mouse and human organoid models of SCN2A deficiency, and acute systemic administration of GABA_A receptor-positive allosteric modulators restores sociability.

 

 

 

Because reduced GABAergic signaling can enhance the E/I ratio and contribute to in vivo neuronal hyperexcitability, we examined whether potentiating GABA_A receptor activity using a positive allosteric modulator (PAM) could normalize neuronal firing. Notably, clonazepam, an FDA-approved benzodiazepine has been shown to rescue social deficits in both a Scn1a knockout model of Dravet syndrome37 and the BTBR model of idiopathic autism38. In WT mice, baseline recordings from putative MSNs showed low firing rates that remained unchanged following acute systemic administration of a low dose of clonazepam (Clz, 0.05 mg/kg, i.p.) (Figure 5C, D). In contrast, clonazepam markedly suppressed the abnormally high firing rates in HOM mice (Figure 5E, F).

 

In summary, our findings reveal that severe Na_V1.2 deficiency produces profound and reversible social deficits, underpinned by disproportionate reductions in excitatory and inhibitory synaptic transmission. We demonstrate a direct, dose-dependent relationship between Scn2a expression and sociability, whereby a ∼70% reduction in Na_V1.2 leads to an elevated E/I ratio, increased noisy basal activity, and impaired neuronal coding, while restoration of Scn2a or pharmacological enhancement of GABA_A receptor function reverses these deficits. Collectively, our work provides a comprehensive exploration, from molecular and cellular mechanisms to neural circuits, of the pathophysiology underlying social impairments in SCN2A-associated disorders. These insights lay a robust foundation for the development of targeted therapeutic interventions aimed at normalizing synaptic function to ameliorate social impairments.

 

The original papers from Professor Catterall in a model of polygenic autism and in Dravet syndrome:

 

Enhancement of Inhibitory Neurotransmission by GABA_A Receptors Having α_2,3-Subunits Ameliorates Behavioral Deficits in a Mouse Model of Autism

Autism spectrum disorder (ASD) may arise from increased ratio of excitatory to inhibitory neurotransmission in the brain. Many pharmacological treatments have been tested in ASD, but only limited success has been achieved. Here we report that BTBR T⁺ Itpr3^tf/J (BTBR) mice, a model of idiopathic autism, have reduced spontaneous GABAergic neurotransmission. Treatment with low non-sedating/non-anxiolytic doses of benzodiazepines, which increase inhibitory neurotransmission through positive allosteric modulation of postsynaptic GABA_A receptors, improved deficits in social interaction, repetitive behavior, and spatial learning. Moreover, negative allosteric modulation of GABA_A receptors impaired social behavior in C57BL/6J and 129SvJ wild-type mice, suggesting reduced inhibitory neurotransmission may contribute to social and cognitive deficits. The dramatic behavioral improvement after low-dose benzodiazepine treatment was subunit-specific—the α_2,3-subunit-selective positive allosteric modulator L-838,417 was effective, but the α₁-subunit-selective drug zolpidem exacerbated social deficits. Impaired GABAergic neurotransmission may contribute to ASD, and α_2,3-subunit-selective positive GABA_A receptor modulation may be an effective treatment.

 

 

Autistic behavior in Scn1a^+/− mice and rescue by enhanced GABAergic transmission

Haploinsufficiency of the SCN1A gene encoding voltage-gated sodium channel Na_V1.1 causes Dravet Syndrome (DS), a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit, and autism-spectrum behaviors. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviors in DS are poorly understood. Here we show that mice with Scn1a haploinsufficiency display hyperactivity, stereotyped behaviors, social interaction deficits, and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odors and social odors are aversive to Scn1a^+/− mice. GABAergic neurotransmission is specifically impaired by this mutation, and selective deletion of Na_V1.1 channels in forebrain interneurons is sufficient to cause these behavioral and cognitive impairments. Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABA_A receptors, completely rescued the abnormal social behaviors and deficits in fear memory in DS mice, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for Na_V1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviors in DS.

 

The 2021 paper from China using the maternal immune activation model of autism:

  

Clonazepam attenuates neurobehavioral abnormalities in offspring exposed to maternal immune activation by enhancing GABAergic neurotransmission

Ample evidence indicates that maternal immune activation (MIA) during gestation is linked to an increased risk for neurodevelopmental and psychiatric disorders, such as autism spectrum disorder (ASD), anxiety and depression, in offspring. However, the underlying mechanism for such a link remains largely elusive. Here, we performed RNA sequencing (RNA-seq) to examine the transcriptional profiles changes in mice in response to MIA and identified that the expression of Scn1a gene, encoding the pore-forming α-subunit of the brain voltage-gated sodium channel type-1 (Na_V1.1) primarily in fast-spiking inhibitory interneurons, was significantly decreased in the medial prefrontal cortex (mPFC) of juvenile offspring after MIA. Moreover, diminished excitatory drive onto interneurons causes reduction of spontaneous gamma-aminobutyric acid (GABA)ergic neurotransmission in the mPFC of MIA offspring, leading to hyperactivity in this brain region. Remarkably, treatment with low-dose benzodiazepines clonazepam, an agonist of GABA_A receptors, completely prevented the behavioral abnormalities, including stereotypies, social deficits, anxiety- and depression-like behavior, via increasing inhibitory neurotransmission as well as decreasing neural activity in the mPFC of MIA offspring. Our results demonstrate that decreased expression of Na_V1.1 in the mPFC leads to abnormalities in maternal inflammation-related behaviors and provides a potential therapeutic strategy for the abnormal behavioral phenotypes observed in the offspring exposed to MIA.

 

Conclusion

On the one hand, it is great that you can use this published research to treat your own child, but it is rather sad that this research is never going to be applied widely to children with severe autism.

Professor Catterall did not want to take on the massive task of “commercializing” his discovery. It would be a huge and expensive job, with no financial return, since clonazepam is already a widely available cheap generic drug.

This is the same problem faced by bumetanide, leucovorin and other generic drugs that can be repurposed for autism.

You have to adjust to the imperfect world we live in, rather than assume everything is being done on your child’s behalf, by those thousands of autism researchers. They want to get published and get paid — that is their success. For me, what matters is getting results, and I did. Hopefully, so will you.