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Tuesday, 23 February 2016

Therapeutic Epigenetics in Autism and Junk DNA




Today’s post takes another dip into the genetics of autism and currently existing therapies that could be re-purposed for autism.  We also see that many secrets remain beyond the 3% of your DNA that usually gets all the research attention.  The remaining 97% is not junk after all.

There was an earlier post on this blog that introduced Epigenetics.  It is not such a complicated subject, just think about it as little tags on your DNA that turn genes on/off usually when they should not be, but there remains the possibility to use epigenetics for good.  In people with under-expression of an important gene you could “tag it” and then increase its expression.

The exome is the part of your DNA that encodes the various proteins needed to build your body.  The remaining 97% of your DNA was once thought to be just junk; we saw in recent post that one part contains enhancers and silencers that control expression of the genes in the 3% that is the exome.

A recent study of gene expression in neurological conditions including autism showed just how broadly disturbed gene expression is.







(A) Consistent fold enrichments were found for each cell type across fourteen cortical and three subcortical brain regions of Alzheimer's patients. The box plots mark the distribution of cellular fold enrichments across all the brain regions examined. Asterisks mark that the fold enrichment for each cell type that was found to be significantly non-zero with p < 0.05. (B) Two independent autism studies show the same cellular phenotypes, including upregulation of glial cells and downregulation of neurons. Asterisks mark those cell types found to be significantly differential with p < 0.05 after BH correction over all groups.


Here I am making the point that even though only a handful of genes may have an identifiable dysfunction, a much broader range of genes seem to be affected, as we see in the wide range of over and under expressed genes.

While it would be logical to think about a specific dysfunction needing a therapy that targets just that gene, this appears not to be necessary.

It appears that downstream processes may be the most damaging/relevant, for example disturbances in Protein Kinase A and C (PKA and PKC) may play a key role in many cases of regressive autism, and this will feature in its own post, because it would be treatable today. 

Reduced activity of protein kinase C in the frontal cortex of subjects with regressive autism: relationship with developmental abnormalities.


Brain Region–Specific Decrease in the Activity and Expression of Protein Kinase A inthe Frontal Cortex of Regressive Autism

 

Both the above papers are by Abha and Ved Chauhan.  I put Abha on my Dean’s list long ago.  I did have a discussion with her a while back.  She is clearly a very nice person and intellectually towers over the Curemark lady (Joan Fallon) who gets $40 million to play with her pancreatic enzymes, but never publishes anything except very superficial patents.


I think for $40 million Abha and Ved could figure it all out.

PKB, otherwise known as Akt is also very relevant to some types of autism.

Tamoxifen, recently shown to reverse autism in a SHANK3 mouse model, is a PKC inhibitor.

Another epigenetic drug, Theophylline activates PKA.

Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer.

If you could identify if a particular person was hypo/hyper in PKA, PKB and PKC, this might well open the door to an effective treatment.


Research on PKB, also known as AKT

Dysregulation of theIGF-I/PI3K/AKT/mTOR signaling pathway in autism spectrum disorders.




And a paper from the clever Japanese:-



Autism spectrum disorder is a set of neurodevelopmental disorders in terms of prevalence, morbidity and impact to the society, which is characterized by intricate behavioral phenotype and deficits in both social and cognitive functions. The molecular pathogenesis of autism spectrum disorder has not been well understood, however, it seems that PI3K, AKT, and its downstream molecules have crucial roles in the molecular pathogenesis of autism spectrum disorder. The PI3K/AKT signaling pathway plays an important role in the regulation of cell proliferation, differentiation, motility, and protein synthesis. Deregulated PI3K/AKT signaling has also been shown to be associated with the autism spectrum disorder. Discovery of molecular biochemical phenotypes would represent a breakthrough in autism research. This study has provided new insight on the mechanism of the disorder and would open up future opportunity for contributions to understand the pathophysiology


For those who favour dietary intervention:-




  
Based on the above chart curcumin should likely be good for my N=1 case of autism. Time will tell.



Consequences of upstream dysfunctions

So it might be better to consider autism as a disease of wider downstream gene expression, rather than necessarily of “faulty” genes.  Modulating the resulting wider gene expression may be much more realistic than fixing individual genes.

It is certainly plausible that the body has its own protective self-repair mechanism that might be somehow re-energized. Some people have pondered why so many highly intelligent mathematicians and computer scientists seem have relatives with autism.  The clever genes do associate with a type of autism plus ID/MR.  It was suggested that protective genetic changes might be in play, so that the people with the most genetic variance are actually the family members without the autism.

This does remain conjecture, but as more whole genome data is collected we are seeing some interesting findings.

A fascinating very recent study that looked at a group of 53 families with autism using the traditional approach of whole exome sequencing and also microarray. 

Using these methods, that are the current gold standard, the researchers found very little.  Dysfunctions in the 700 known autism genes were not detected.

However using more expensive whole genome sequencing, dysfunctions were identified in the “DNA junk” zone very close beside the known autism genes.  The researchers were then able to identify the genetic cause of 30% of the cases, a big improvement on 0%.  I expect if they looked a little harder the 30% would be higher.


“We performed whole-genome sequencing (WGS) of 208 genomes from 53 families affected by simplex autism.”

“For the majority of these families, no copy-number variant (CNV) or candidate de novo gene-disruptive single-nucleotide variant (SNV) had been detected by microarray or whole-exome sequencing (WES).

Comparing the sequences of the individuals with autism and those of their unaffected siblings, the researchers found that people with autism are more likely to have genetic variants — either single base-pair changes in the sequence or small CNVs — in swaths of DNA abutting known autism genes. But the researchers only found the variants after they restricted their search to regions of the genome already implicated in autism, and even then the statistical significance is modest.

Sequencing whole genomes could reveal the genetic cause of autism in as much as 30 percent of people for whom faster and cheaper sequencing methods come up short

“It’s increasing power even in areas that are supposed to be covered by whole-exome sequencing,” says Peixoto. “It seems that it’s clear that whole-genome sequencing will become the standard.”







One specific microRNA has strong links to autism spectrum disorder, say TSRI scientists


Epigenopathies

Many diseases have an epigenetic component. The severe progressive asthma that is COPD is a well-known example.  It appears that smoking in middle age often leads to permanent epigenetic changes that come back to haunt often then non-smokers in old age.  Even though they have not smoked for twenty years, there oxidative stress response has been permanently modified.  This results in a kind of steroid resistance, so that usually reliable drug therapies fail to work. 

It is thought that autism has an epigenetic component.  This would do some way to explaining 30-40% of the increase in prevalence in recent years that is not explained by ever widening diagnostic criteria.

Because epigenetic changes can be heritable and can be accumulated from all kinds of exposures, even simple ones like severe emotional stress and pollution, you can reconcile autism as being primarily a genetic condition even though incidence has clearly risen within one or two generations. So you can have an “epigenetic epidemic”, so to speak.


Epigenetics as a therapy

While much is written about epigenetic change being bad, it could also be good.

There are many known substances that affect gene expression; some are very target specific which is useful.

This answers a recent issue raised by a reader of this blog who did exome sequencing. What is the point of discovering a genetic dysfunction if there is no therapy? Medicine is some decades behind science, better to know what gene is affected because you well be able to affect its expression, you just need some help from Google.

Epigenetic therapy could be used to remove unwanted tags, but it could also be used to leave new ones to upregulate under-expressed genes.

Such epigenetic therapy is already a reality in COPD and is being considered for rare single autisms where one copy of the gene is not functional, so turn up the volume on the remaining copy.

As we saw in the post on epigenetics, one potential category of drugs are HDAC inhibitors, these would affect one epigenetic mechanism.

There are many such HDAC inhibitors and most have other modes of action, so you cannot be sure what is giving the noted effect.


Valproate

This epilepsy drug has numerous effects including as a HDAC inhibitor.  Given to mothers during pregnancy it can cause autism in the offspring, but when given to the affected offspring the autism can be reduced.

Valproate is given off label to treat autism even when no epilepsy is present.

As we saw in the comments section, long term valproate se can have side effects.


Sulforaphane

This substance derived from broccoli and patented by Johns Hopkins, is another HDAC inhibitor.  It also upregulates Nrf2, which turns on the oxidative response genes.  This was proposed as a COPD therapy by Professor Barnes.

We saw in a post that for Nrf2 to have its full effect there needed to be enough of a protein called DJ-1.  You can increase DJ-1 expression with cinnamon (sodium benzoate).

That was one reason to think that cinnamon would complement Sulforaphane as a therapy for both COPD and some autism.


Sodium Butyrate

Sodium Butyrate is an HDAC inhibitor that is available as a supplement. We came across it in an earlier post as a precursor to butyric acid.  Butyric acid plays a role in the permeability of the gut and the Blood Brain Barrier (BBB).  It also seems to protect from auto immune disease.

Butyrate is fed to millions of farm animals every day to increase their resistance to auto-immune disease.

Butyric acid is produced naturally in the gut by the bacteria living there, however the amount can be increased by the uses of a particular probiotic-bacteria.

This would support the uses of sodium butyrate and the Miyari 588 bacteria.

I have on my to-do-list to investigate higher doses of Miyari 588, but having read the comment by Alli that 500 mg of sodium butyrate is effective, I will try that first.  She also found higher doses ineffective, which was the same in a mouse study published last November,

The study below highlights which genes were down-regulated and which were up-regulated, the overall effect was beneficial


Sodium butyrate attenuate ssocial behavior deficits and modifies the transcription ofinhibitory/excitatory genes in the frontal cortex of an autism model.

 

The core behavioral symptoms of Autism Spectrum Disorders (ASD) include dysregulation of social communication and the presence of repetitive behaviors. However, there is no pharmacological agent that is currently used to target these core symptoms. Epigenetic dysregulation has been implicated in the etiology of ASD, and may present a pharmacological target. The effect of sodium butyrate, a histone deacetylase inhibitor, on social behavior and repetitive behavior, and the frontal cortex transcriptome, was examined in the BTBR autism mouse model. A 100 mg/kg dose, but not a 1200 mg/kg dose, of sodium butyrate attenuated social deficits in the BTBR mouse model. In addition, both doses decreased marble burying, an indication of repetitive behavior, but had no significant effect on self-grooming. Using RNA-seq, we determined that the 100 mg/kg dose of sodium butyrate induced changes in many behavior-related genes in the prefrontal cortex, and particularly affected genes involved in neuronal excitation or inhibition. The decrease in several excitatory neurotransmitter and neuronal activation marker genes, including cFos Grin2b, and Adra1, together with the increase in inhibitory neurotransmitter genes Drd2 and Gabrg1, suggests that sodium butyrate promotes the transcription of inhibitory pathway transcripts. Finally, DMCM, a GABA reverse agonist, decreased social behaviors in sodium butyrate treated BTBR mice, suggesting that sodium butyrate increases social behaviors through modulation of the excitatory/inhibitory balance. Therefore, transcriptional modulation by sodium butyrate may have beneficial effects on autism related behaviors.


  

Theophylline

Theophylline is an old asthma drug that is an HDAC inhibitor.

At low doses it is now being trialled as an epigenetic add-on therapy in COPD.  It pretty obviously does work, but data needs to be collected to measure how effective it is and what is the best dose.

It shows how the COPD researchers/clinicians like Professor Barnes are doing a good job and not frightened to experiment.

Would a similar low dose of theophylline benefit a sub-group of those with autism/schizophrenia?  I think it is quite likely.

COPD and autism/schizophrenia share the same impaired oxidative stress response.



Chronic Obstructive Pulmonary Disease (COPD) is a progressive lung disease characterised by progressive airflow limitation. In the UK, it affects around 3 million people, is the fifth leading cause of death and costs the NHS approximately £1 billion annually. Exacerbations of COPD account for 60% of NHS COPD costs and are associated with accelerated rate of lung function decline, reduced physical activity, reduced quality of life, increased mortality and increased risk of co-morbidities. COPD treatment guidelines recommend inhaled corticosteroids (ICS) to reduce exacerbations and improve lung function. However, in COPD, airway inflammation is relatively insensitive to the anti-inflammatory effects of ICS and even high doses fail to prevent exacerbations. Preclinical and pilot studies demonstrate that low dose theophylline may increase the sensitivity of the airway inflammation to ICS, and thus when used with ICS will reduce the rate of COPD exacerbation. In this study we will determine the clinical effectiveness and cost-effectiveness of adding low dose theophylline to ICS therapy in patients with COPD. The primary outcome is the number of exacerbations. The primary economic outcome is the cost-per-QALY gained during the one year treatment period. We will recruit 1424 participants from primary and secondary care across seven areas of the UK. Participants will be randomised to theophylline (200 mg once or twice daily depending on smoking status and weight) or placebo for 12 months. We will follow participants up at six and twelve months to assess the number of exacerbations. We will also collect data on adverse events, health care utilisation, quality of life and breathlessness, and lung function. Low dose theophylline is cheap (10p/day) and, if shown to make current ICS therapy more effective in a cost effective manner, it will improve the quality of life of COPD patients and reduce the burden of COPD on the NHS.


At large doses, Theophylline has long been a therapy for asthma and COPD, but as with Sodium Butyrate, it is quite possible that larger doses of Theophylline produce a different result.  In other words the epigenetic effect fortunately comes from the low dose.

Low doses mean less chance of side effects.

For example, in anyone predisposed to reflux/GERD/GORD many asthma drugs pose a problem because at the same time as opening the airways in your lungs they will relax the lower esophageal sphincter and allow stomach acid to rise upwards.

We saw in an earlier post that in some types of autism something called mGluR5 is dysfunctional in the brain. By chance mGluR5 is also involved in closing the lower esophageal sphincter.  In people with reflux/GERD/GORD a mGluR5 inhibitor was found to have promise for the management of their symptoms.


Randomised clinical trial:effects of monotherapy with ADX10059, a mGluR5 inhibitor, on symptoms and reflux events in patients with gastro-oesophageal reflux disease.




So it is not surprising that many people with autism also have reflux/GERD/GORD. 

But the dysfunction with mGluR5 in autism can be both hyper and hypo, so the therapy might be a positive allosteric modulator (PAM), or a negative allosteric modulator (NAM).  

In someone with autism + reflux/GERD/GORD  it would be reasonable to think a NAM, like ADX10059, might help both conditions.



Gene Repression and Genome Stability

There is another epigenetic process that may be disturbing gene expression in some people and may be treatable.

I have been trying to find why so many people with autism can benefit from biotin; I think I have found a plausible explanation.

“Biotinylation of histones plays a role in gene repression and repression of transposable elements, thereby maintaining genome stability”

I think in some people with autism and no clinical deficiency of biotin the continued “overdosing” of biotin might be having an effect on gene expression, bringing things a little closer to where they should be.

Rather beyond the scope of this blog, it appears that in some people the impaired genome stability, reversible with biotin(ylation), this might be a significant cancer risk.

In essence, for most people supraphysiological concentrations of biotin will do absolutely nothing, but in a sub-group it might do a lot of good.  It is epigenetic, but you do not have to understand it to benefit from it.  It is complicated.




Transposable elements such as long terminal repeats (LTR) constitute 45% of the human genome; transposition events impair genome stability. Fifty-four promoter-active retrotransposons have been identified in humans. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events, and abnormal regulation of genes. Here, we demonstrate that the covalent binding of the vitamin biotin to lysine-12 in histone H4 (H4K12bio) and lysine-9 in histone H2A (H2AK9bio), mediated by holocarboxylase synthetase (HCS), is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of H4K12bio and H2AK9bio at intact retrotransposons and a solitary LTR depended on biotin supply and HCS activity and was inversely linked with the abundance of LTR transcripts. Knockdown of HCS in Drosophila melanogaster enhances retrotransposition in the germline. Importantly, we demonstrated that depletion of H4K12bio and H2AK9bio in biotin-deficient cells correlates with increased production of viral particles and transposition events and ultimately decreases chromosomal stability. Collectively, this study reveals a novel diet-dependent epigenetic mechanism that could affect cancer risk.

Here, we provide evidence for the existence of a novel diet-dependent epigenetic mechanism that represses retrotransposons. Importantly, we demonstrated that depletion of biotinylated histones in biotin-deficient cells increases LTR transcript levels, production of viral particles, and retrotransposition events, and ultimately decreases chromosomal stability. Both biotin deficiency and supplementation are prevalent in the US. For example, moderate biotin deficiency has been observed in up to 50% of pregnant women (35,36). About 20% of the US population reports taking biotin supplements (37), producing supraphysiological concentrations of vitamin in tissues and body fluids (23,28,35). The findings presented here suggest that altered biotin status in these population subgroups might affect chromosomal stability and cancer risk. 

Biotin and biotinidase deficiency


Biotin requirements for DNA damage prevention



  

Conclusion

I never got round to writing part 2 of my epigenetics post, but my experience of HDAC inhibitors to date has been very positive.

I would be the first to admit that this is rather hit and miss.  It was only when reading the paper on potential therapies for Pitt Hopkins, that was openly musing about HDAC inhibitors, in an equally hit and miss approach, that I thought I would write further about it.

It really seems totally haphazard, because you cannot predict the effect with any level of certainty.  If there is a self-repair mechanism trying to maintain homeostasis of the genome, haphazard may be good enough.

10mg of biotin twice a day does have a mild but noticeable stabilizing effect; is this caused by better maintaining genome stability? I have no idea. 

I will try sodium butyrate and if it works I will have to establish what dose of Miyari 588 produces the same effect.  Both are used in animal feed to reduce inflammatory disease, so you are already indirectly exposed to them if you eat meat.

Theophylline should also be investigated.  This is a very well understood drug and small doses really do seem to help people with COPD.

PKA, PKB and PKC are likely at the core of most people’s autism.  Many existing therapies can modify their expression.

Whole genome sequencing, carried out at great precision, is clearly the only satisfactory genetic testing method.  The other, cheaper, methods are just missing key data and giving many false negative results, i.e. saying there are no identifiable genetic dysfunctions, when this is not true.







34 comments:

  1. My son has autoimmune issues and has been noted that his Histone Autoantibodies are high which is sometime indicative of lupus or drug induced lupus and they are monitoring this marker (which he doesn't have lupus). I don't think many autistic kids get tested for this. I see sodium butyrate is a histone deacetylase inhibitor and biotin is has impacted too. What is the connection of a histone deacetylase inhibitor, for example, and high histone autoantibodies which are not good. Lots of good food for thought.

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  2. I would be careful with going overboard on the curcumin though, as it seems to be a strong inducer of the p53 pathway which could lead to excessive apoptosis of healthy cells. Really, the vast majority of research on curcumin is positive but there is some research that one of its mechanisms of action is through causing DNA damage, which of course upregulates the stress response pathways to fix the DNA damage (i.e. a hormetic effect).

    With regards to AKT, there is also good old metformin which has many similarities to curcumin as well.

    Another alternative is to go plain old turmeric because there are other compounds in turmeric such as turmerone which seem to be relevant to improving outcomes in brain damaged diseases (which one could argue includes autism):

    http://www.eurekalert.org/pub_releases/2014-09/bc-tcb092314.php

    With respect to HDAC inhibitors, I mentioned before that one of the best ways of boosting butyrate levels is to simply supplement soluble fiber into the diet. I personally use a combination of inulin, barley flour, and potato starch, but there are other alternatives and which form of soluble fiber is best is a matter of argument, though in my opinion variety is best since different species of gut bacteria utilize different forms of soluble fiber differently and for reasons researchers are not sure of yet, it appears gut microbiota complexity is a hallmark of good health and also happens to be severely diminished in autism.

    As to Anonymous' question, coincidentally I have read more than I care to share about polyamines (with respect to autism therapies) and one thing that is frequently mentioned in papers is that polyamines are raised in autoimmune disorders. Reasons for them being elevated range from the "leaky gut" hypothesis where bacteria produce excess polyamines that leak into the body, to the "hygiene hypothesis" where parasitic worms can help modulate hyperimmunity possibly by gobbling up excess polyamines in the gut.

    At the moment I don't think polyamines are good or bad necessarily (the body tries to tightly control their levels), but I think temporary boosts of various ones have potential in some therapies, while in the case of your son you may want to limit polyamine rich foods and other substances which upregulate ornithine decarboxylase which is the enzyme that converts ornithine to putrescine (ornithine is derived from arginine so if you want to lower arginine levels, supplement L-Lysine which competes with arginine for absorption).

    On the other hand, I think in many autistic cases there are problems with not enough arginine in the brain (just a personal hypothesis based on recent Alzeheimer's research), so one way of preventing arginine from being converted to ornithine is to increase putrescine levels because it downregulates the enzyme that causes arginine to be converted into putrescine.

    Orange juice and grapefruit juice have a lot of putrescine so you may want to avoid them, or maybe try giving your son significant quantities of putrescine as the temporary boost might have a paradoxical response of normalizing polyamine levels in an autoimmune disorder by downregulating the enzymes that convert ornithine to putrescine (just speculating here).

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  3. Peter, in your article you say that if we can identify a hypo/hyper in kinase, we might find an effective treatment. According to my son's recent biochemical tests, protein kinase C seems quite low. He is 67,0 U/L and normal means are 25-200. Is this a reliable indication?
    If he is found low, would fish oil help? Lots of them come with DHA and EPA. What about green tea? What other interventions would be worth a trial?
    I am trialling high doses of vitamin c (2 gr/day) and it never occured to me such a simple intervention would raise his energy/cognitive levels so surprisingly "high".
    Thank you

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    Replies
    1. Petra, I think you would need to know the protein kinase C level in the brain. This may well be very different to the level in the blood.

      Low levels of PKC and PKA were found in regressive autism. So I think this would likely not be the case in your son. But anything is possible.

      Since you had luck with NAC and now another antioxidant, vitamin C, I would focus on antioxidants (oxidative stress). There are various forms of Vitamin E that also seem to help some people, again it is a potent antioxidant.

      If your son is avoiding fruit and vegetables he may have low levels of many substances.

      I think eating fish is better than supplements. Olive oil is also good, but you probably consume a lot of that.

      Delete
  4. Berberine looks very promising for activating AMPK as well as helping with many issues relevant to autism, such as gut, inflammation, mental health, lipids metabolism and so on. Would you consider approving it?

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    Replies
    1. Petra, berberine does indeed have interesting properties. For people with type 2 diabetes it really does seem to help. Whether it will help your son, I do not know.

      If your son has oxidative stress and L-carnitine also helps (implicating mitochondria), I would try things like “Life Extension Gamma E Tocopherol-Tocotrienols". This is an OTC supplement that is almost the same as EPI-743, being developed for mitochondrial disease. It is just a modified vitamin E supplement, so it will either help or do nothing.

      Delete
    2. Hello Peter. Thanks for your advice on trialling other possible antioxidants.
      Peter I have to admit that I had a 'miss' if this is the word that describes it. I've already mentioned that with carnitine things got really better for my son. After almost 25 days of administrating 2grms of carnitine, his anxiety levels raised and my son had a meltdown. He didn't have a meltdown from early November when he started taking Nac. The meltdown was quite mild. As I have been warned from Nat about carnitine's possible side effects on behaviours, I suspect this is responsible for his regression. Today I am going to stop it and hope that it gets out of his system.
      As I need to address to his anxiety issues, I can either trial baclofen or vitamin A to upregulate oxytocin leves.
      What would be an initial effective dose of baclofen? I need to take it easy. As for oxytocin, trialling vitamin A looks simple, but what about other oxytocin interventions, like optinose for example, do we have any feedback on that? Do other oxytocin drugs you can buy at the chemist's seem to work?
      Thank you

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    3. Petra, the UK pediatrician who commented on this blog about her/his success treating Asperger's with Baclofen did not give all the details. In the UK doctors are now very cautious about any off-label prescribing.

      Baclofen is normally given three times a day. That doctor said there was no problem will side effects or tolerance and about 70% were responders - that is excellent result. The smallest pill is 10mg, but I do not know what dose was used. It would be good to connect that doctor with your "biotin" doctor, but I do not have the contact details. Autism is not supposed to be treatable and so most doctors do not openly discuss doing so.

      Normally it takes 1 or 2 months to get the effect of Baclofen, in what it is usually prescribed for. I think you will see the "Asperger's effect" much faster.

      I really would suggest you ask your new doctor, who seems very open minded about autism. You could also ask him about syntocinon, nasal spray, which is the oxytocin nasal spray prescription drug.

      Delete
  5. Is whole genome sequencing available without being part of a study and if so, are there certain companies and/or types of sequencing more accurate/effective than others? Lastly, I am wondering about cost....
    Thanks
    Nancy

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    Replies
    1. Nancy, whole genome sequencing is available without being part of a study. It costs about $ 1,500 per genome and they like to do the parents as well. The caveat is that this is usually not a clinical service but for research purposes.

      So it is easy to spend lots of money. The quality will vary because only when you look in great detail do you find the anomalies. The real value is in the analysis and in commercial services for Exome sequencing this can be poor, and then it is a waste of money.

      Some researchers are very nice people and if you want to pursue this, I would contact the authors of the study in my post (Department of Genome Sciences, University of Washington School of Medicine, Seattle) and offer your son "for research purposes".

      They might be very helpful, particularly if you refer their own recent paper. So it might not cost anything.

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    2. Peter, there is another article regarding ASD genetic "signature" that kind of confirms the one you mention in this post (and most of what you have been publishing actually).

      http://www.nature.com/tp/journal/v6/n1/full/tp2015112a.html

      I imagine that once/if/when this is confirmed, genetic testing will be more useful to guide treatment, or at the very least allow detection at young age.

      J.

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  6. Peter,

    As usual you have provided an insightful analysis that goes well beyond what most autism researchers are able to synthesize.

    I think your comments regarding gene stability and biotin are interesting. I have wondered about biotin as well. I believe it is also involved in fatty acid metabolism and a number of people with autism have fatty acid metabolism inefficiencies.

    Biotin is also one of the sulfur containing B vitamins and sulfur deficits seem to characterize autism. This statement may sound simplistic but the other sulfur containing B vitamin is thiamine which is also often deficient in autism. There is a particular form of thiamine called benfotiamine that is lipid soluble. It is often used in treating diabetic neuropathy, a disease of the nerves that is tied to inflammation and oxidative stress. You could make the case that autism is typically another disease of nerve inflammation and oxidative stress. So one would think benfotiamine may be effective in autism. In experiments with my son, I feel that benfotiamine supplementation does indeed result in significant improvement in mood and functional level. I would be quite interested in the experience that others' have with it. I would guess that the therapeutic value of benfotiamine in my son would be replicated in most others with autism since typical autism biochemistry seems to have significant overlap with low thiamine biochemistry and benfotiamine seems to have better bioavailability than water soluble forms.

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    Replies
    1. Seth, what dosage of benfotiamine do you use?

      It is very interesting just how many substances that help type 1 or type 2 diabetes also help autism. It is a long list and at least with diabetes the effect is very obvious and in the case of insulin sensitivity is directly measurable (less insulin is needed).

      If you want to write another Guest Post on the benfotiamine I think that would generate the most impact.

      Biotin does help in a substantial minority of cases.

      Again it is remarkable how many B vitamins can help the right sub-group. B12 seems to help about 15%. B9 (folate) in large doses also helps a sub-group.

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    2. Hi Seth,

      My daughter was on 80mg of benfotiamine for a while, no visible effects. None of the B vitamins seem to do anything for her, we did the very high dose b12 shots, biotin 8mg, b6 made her hyper, so we did p5p with magnesium at 50mg and then 100mg. Nothing. Finally, stopped them all.

      Delete
    3. Hi,

      I tried benfotiamine after I read a paper suggesting its effect in microglial activation:
      http://www.ncbi.nlm.nih.gov/pubmed/26388737

      "benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus."

      And another one from the same team:
      http://www.ncbi.nlm.nih.gov/pubmed/26388737

      I tried it only for short time as it turned out to be one of those substances which apparently have positive effect in my son, but are associated with hyperactivity. Carnitine is another example.

      Peter, I can't find the post about my mirtazapine experience, so I continue here. As I wrote previously the day 1. was bad for my son and I quit, but then from day 2. to 4. we saw a clear improvement with imagination, speech and something which is probably called 'executive functioning'. Then in 2 days he developed really high fever and his infection is ongoing until now. I've heard that some people experience fever effect even before fever occurs, on the other hand if mirtazapine metabolism is indeed slowed by CYP3A4 inhibition, then it's blood level might be somewhat unpredictable. T1/2 is 20-40 hours so I wonder if it might be mirtazapine decreased levels effect in next days? What do you think? Did you try mirtazapine?

      Delete
    4. Hi Peter,

      I am using 120 mg of benfotiamine per day currently with my son. He is about 80 pounds. Regarding methylcobalamin and methylfolate, my son does better on them too. He also has high methylmalonic acid and forminoglutamic acid in his urine if unsupplemented. These are indicators of deficency as you likely know.

      RG,

      Thank you so much for the color regarding your experience with benfotiamine with your daughter. I am sorry to hear that it did not help her but I would rather know about its affects in others before I push to hard about its relevance in many cases of autism. I think sharing things that did not work are as important as sharing things that did.

      Regarding your daughter, do you by chance have the resources available to get a quantitative organic acid test done by Great Plains Labs or Genova Diagnostics? If there is something metabolic, a good quantitative organic acid test is likely to pick it up because they measure a large number of metabolites.

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    5. Agnieszka, I have not yet tried mirtazapine.

      In many trials they use very high doses and they risk missing the possible good effect of low doses. For example one reader found 500mg of sodium butyrate beneficial, but not higher doses. A clinical trial is planned to trial sodium butyrate to improve cognitive function in schizophrenia, the dose is 6.5g.

      Perhaps try mirtazapine again but with an even smaller dose?

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    6. Hi Seth,

      we did the Genova organic acid test, and it indicated deficiency for all B vitamins, specially B9 and B6, it was the only point of concern in his results. At the time we tried a B Complex as suggested by our DAN "consultant" (a naturopath, not MD), with awful results. I've ended up abandoning DAN for other reasons.

      I decided lately to try individual B vitamins instead of the complex, to know what works and what doesn't.

      I've started P5P with no issues but no noticeable benefit.

      Then I trialed calcium folinate, and he had an amazing day for cognition, followed by awful motor regression and dyskinesia. I suspended it right away, but it took about 3 weeks for the side effects to fade.
      Since day 1 was good, I decided to try 5-MTHF instead, as I suspected the calcium from the other formulation could be the issue. Its less stellar on the good side, but without side effects, should be a win, I will observe and decide in the next weeks.

      B12 will be next, but "Methylcobalamin is not sufficient as a singular source of vitamin B12", as "is not converted into adenosylcobalamin".
      "Defficiency of adenosylcobalamin disturbs carbohydrate, fat and amino-acid metabolism" (interesting!)
      https://en.wikipedia.org/wiki/Methylcobalamin

      Regards,

      J.





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    7. Hi Seth & RG,
      I stumbled on your post and we did have a similar experience. Our DAN ordered the Genova diagnostic test - nutrieval and we got that his b vitamins were severely lacking as well as pyruvic acid and butyric acid were really high (mitochondrial dysfunction), but when our DAN gave us a compounded prescription, it has made our son really hyper, (stimming more) and we stopped it.
      We were also wondering if we should do B12 shots? we do drops now. Would appreciate any input
      Thanks
      BK

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    8. BK and RG,

      I am sorry to hear that the results were not favorable. I have been there too. It was suggested that we give our son oral vitamin D because of low levels of 25-hydroxy vitamin D, which was a very bad move in his case. The low levels can be indicators of inflammation. I think doctors fall into the trap of seeing low levels of something as an indication that supplementation is desirable and they often give too much as they are going for a "therapeutic" dose. In my opinion one should only consider supplementing a subset of nutrients which seem to have some relevance to what is common in autism biochemistry - not saying this approach will avoid all pitfalls.

      Regarding B12, I would not do shots. Medical literature suggests oral B12 is absorbed. If you are concerned he is not getting enough, then you can give more.

      More generally I think it makes a lot of sense to try things that have worked for others with autism. While nobody is the same there is often commonality in biochemistry.

      RG, I like your idea of methylfolate and cobalamin for this reason. As Peter mentioned biotin has some interesting properties too if you are restricting yourself to B vitamins. I think elevations in valeric acids are indicative of possible deficiency. Another route you might go is look for antioxidants that might help if there is high ox stress. I know Peter has a lot of good ideas on this. Carnitine helps a subset of kids and can serve as an antioxidant. Anonymous, if pyruvic acid is high, this can be a sign of mitochondrial dysfunction and carnitine might be helpful - admittedly it might not be helpful too. Benfotiamine can also help with mitochondrial related issues. Separately, I have found carotenoids (antixidants) to help my son some but there are no trials in the literature to my knowledge. NAC is also an antioxidant and helps some kids but not my son. When trying something always try low doses first to lower risk.

      As you know Peter has a lot of great ideas around the pharmaceutical angle also which I know less about.

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    9. Hi BK,

      I second Seth on not doing the shots. Apparently, in spite of refrigeration, there are issues with breakdown. The lozenges are equally, if not more, effective. My husband has been taking the Jarrow's 5000mg lozenges, and his levels on testing have been very good, in fact, a bit too much, and he has cut down.

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    10. Hi Seth,

      I did do the OAT several years ago, and it came back normal. I have always had my doubts about these tests, particularly because I wonder where they get their reference range from. I am also not a believer in the yeast theory. It appears that treating to those tests is a 50-50 chance, which means you don't need the tests at all, but could just try some things and see what works.

      We had an interesting experience with vitamin D. A few years ago, my daughter tested low and supplementing at around 2000u did not do much. She was still at low normal. Then, I went up to 6000iu, intending it to be temporary, just until levels came up adequately. As it happened, she then became very happy, all traces of irritability, distress and mild SIB went away. In fact, once on holiday, she got a double dose by mistake and suddenly for an entire day, we had what appeared to be a non autistic, normal girl. She spoke fluently and appropriately, joined us at breakfast and asked for some coffee, which she had never in her life tasted, went skiing etc. So, the high dose vitamin D stayed. Regular testing showed levels between 60-70. We stayed at this dose for a while. Then, I found that her calcitrol levels were too high. She had also developed horrible plaque that took 3 visits to the dentist to clean. Within a year, it had reformed again. In spite of excellent dental hygiene and not eating sweets. High calcitrol apparently indicates poor calcium absorption, but she was eating yogurt every day. It seemed that supplement K2 might have been necessary. By then the vitamin d had stopped working when other things like epilepsy and raging and SIB had worsened. So I stopped the vitamin D altogether, instead of adding more things that I wouldn't be able to gauge the effect of.

      For things like carnitine, I prefer to use a regular amino acid panel. Both total and free carnitine came back normal, so have not wanted to supplement. In some people, it can make their epilepsy worse. NAC was very good for us until it stopped working due to allergies. I plan on putting it back in.

      Sorry for the long winded comment, just wanted to add to your data set.

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  7. Hello Peter, As I am trying to come up with a logical explanation with carnitine trial failure, I think I overdose it.
    My Aspergers sister, who doesn't look the part, can be very explicit with thoughts and feelings. She took carnitine at noon, forgot about it, then at supper time she felt the benefit of her food so greatly that a positive thought came to her mind and went on with her evening with a feeling of fulfilment.
    The other day she took carnitine together with vitamin C and wanted to bang her head on the wall, although she never does that.
    I didn't know that ascorbic acid participates in carnitine's biosynthesis and possible anxiolytic/anxiogenic effects are dose dependent.
    Maybe I'll try again low dose carnitine (5oo mg/day) before I turn it down?

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    Replies
    1. Petra, it is best to use small doses because often there are multiple modes of action and there are interactions with other therapies. You can always cut pills in half. Maybe 250 mg is plenty

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  8. Hi Peter

    I have been reading your posts for awhile now. My son has a confirmed PTEN mutation that was investigated after me pushing to test further to its normal microarray. My son has macrocephaly and autism. I am still confused about the best course of action; should I stimulate his PTEN and inhibit MTOR?
    Fish oil and acetyl l carninite have been the most helpful for him so far. I want to try curcumin and neuroprotek.
    I know you have been reading a lot about pten and MTOR. Could you suggest something based on your research?
    Thanks in advance!

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    Replies
    1. Hi Laura, under-expression of PTEN will lead to an enlarged brain and head. There will be too much Akt/PKB. This causes further cascades leading, for example, to higher levels of mTOR and NF-kB.

      As you can read in Wikipedia:-“ When defective, PTEN protein interacts with the protein of a second gene known as Tp53 to dampen energy production in neurons. This severe stress leads to a spike in harmful mitochondrial DNA changes and abnormal levels of energy production in the cerebellum and hippocampus, brain regions critical for social behavior and cognition. When PTEN protein is insufficient, its interaction with p53 triggers deficiencies and defects in other proteins that also have been found in patients with learning disabilities including autism.”

      So if you have proof that PTEN is under-expressed, I would focus on increasing PTEN. I would use a serious drug, not a supplement. Statins, normally used to reduce cholesterol, are proven to increase PTEN. You need a lipophilic one such as Lovastatin, Simvastatin or Atorvastatin. You can also try curcumin, but what works in the test tube may not in humans. We use Atorvastatin (Lipitor) 10mg.

      I think you would see the beneficial effect very quickly.

      If it has the desired effect, you just need to also give coenzymeQ10 because this is depleted by the statin. This seems to be the key side effect of statins, but is easily compensated for.

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  9. Hi Peter,
    Thank you so much for your reply!
    Now the challenge will be to find a doctor in Canada that would be willing to prescribe a statin for my 4 year old. Would you happen to have any link to a research/article that talks about the relation between statins and PTEN? Or guide me to find one?
    Thanks again!
    Laura

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    Replies
    1. Laura, there three important issues.

      Does the statin upregulate PTEN ?

      Does upregulated PTEN improve your child’s symptoms? (only way is to try it)

      Is a statin drug safe in a 4 year old?

      I would start with safety. Due to the rise in childhood obesity, the age at which statins are approved for use to lower cholesterol is 10 years old for Atorvastatin at a dose of up to 20mg. Statins have been used to lower cholesterol in even younger children.

      Use of Statins for Dyslipidemia in the Pediatric Population
      http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018249/

      Summary of pediatric studies involving the statins
      http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018249/table/i1551-6776-15-3-160-t03/


      Statins are used for other than to lower cholesterol, for example to treat Neurofibramatosis in children. This is using the statin in people with normal cholesterol. The study referred to below found the statin safe.

      Common cholesterol drug safe, may improve learning disabilities in patients with neurofibromatosis, study finds
      http://www.sciencedaily.com/releases/2011/09/110927124701.htm

      Evidence showing Statins do upregulate PTEN

      Regulation of the PTEN promoter by statins and SREBP
      http://hmg.oxfordjournals.org/content/17/7/919.full.pdf

      “In our current study, we show that simvastatin, pravastatin and fluvastatin can induce PTEN expression in a dosedependent manner. This resulted from an increase in PTEN mRNA indicating transcriptional upregulation. In addition, we observed, for the first time, that upregulation of sterol response element-binding protein (SREBP), known to induce PPARg expression, can increase PTEN expression. “
      “Overall, our observations suggest that statins signal through another transcription factor, in a PPARg-dependent manner, which in turn induces PTEN transcription”

      Inhibitory effect of atorvastatin on the cell growth of cardiac myxomas via the PTEN and PHLPP2 phosphatase signaling pathway.
      http://www.ncbi.nlm.nih.gov/pubmed/23715723

      “atorvastatin may be a potential drug for the treatment of CM (cardiac myxomas) by enhancing the activity of PTEN and PHLPP2


      I am not sure a family doctor would ever prescribe a statin to a 4 year old, even though it may be entirely safe.

      All the various pathways involving PTEN, Akt etc are interrelated and too much PTEN can cause type 2 diabetes. So your doctor may not want to give you the statin. Just like too little PTEN is damaging, so is too much. Personally, I would have thought someone with a PTEN mutation is the last person who needs to worry about excessive PTEN causing diabetes.

      Since your child is very young, Canadian, and you do have supporting genetic evidence I would suggest you contact Dr Clara van Karnebeek at Biochemical Diseases Division of BC Children’s Hospital in Vancouver. http://www.bcchildrens.ca/

      She has both an academic and clinical interest in treatable inborn errors. She has a website (http://www.treatable-id.org/)

      Failing that, you can always find an older relative who has high cholesterol.

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    2. Laura, there is an alternative method to upregulate PTEN and that is to use a potent peroxisome proliferator-activated receptor gamma (PPAR gamma) agonist. These include type 2 diabetes drugs like rosiglitazone but also some natural substances like Salvia Sahendica (containing Daucosterol) from Iran.

      Inactivation of PI3K/Akt pathway and upregulation of PTENgene are involved in daucosterol, isolated from Salvia sahendica, induced apoptosis in human breast adenocarcinoma cells
      http://www.sciencedirect.com/science/article/pii/S0254629914000520

      Drugs like rosiglitazone have far more troubling side effects than statins.

      I still think statins are best. They are more safe and standardized.

      But for other people wanting a mild PPAR gamma agonist that needs no prescription daucosterol may be of interest. Sytrinol (Tangeretin) is another choice for them.

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  10. I can't thank you enough! I will review all this information. Hopefully I can find a way to make this work. I'll keep you posted.

    ReplyDelete
  11. Hi Peter,
    I was wondering if you have any thoughts about cbd oil as an up regulator of PTEN or about its other uses in autism. It is legal where we live and accessible.
    Thanks again for your input!
    Laura

    ReplyDelete
    Replies
    1. I think cbd oil has many effects and really you need a big effect on PTEN and just that any other effects are not problematic. I would ask a specialist like the lady in Vancouver, she may well have a better idea. In our case the effect of the statin was from the first pill.

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  12. We conducted a week long trial of Biogaia Gastrus with our daughter. Although we observed no clear benefit, we have continued to use it as our "resident" probiotic (at only 1 tablet per day) due to it's scientifically verified benefits. We are now interested in trialing Miyairi 588 because of a recent stool analysis that showed no B. Fragilis group in our daughter's gut and a low level of butyrate. Do you have an opinion about whether it is counterproductive to use two different probiotic strains? Since probiotics function in part by crowding other organisms out, I'm wondering if they will work against each other. I would be interested in your opinion about this. Thanks!

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    Replies
    1. We use both Miyairi and Biogaia Protectis. You might also see if giving a butyrate suplement does some good. It is not clear how much Miyairi you need to give to equal say 500mg of butyrate supplement. Butyrate does help some people with autism. Both butyrate and Miyairi improve immune health in animals.

      I suppose there may be an effect by combining probiotics, but we experienced no issues.

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