Bumetanide for TSC-type Autism, Verapamil now for sinusitis, Lower dose Folinic Acid looks interesting for Autism in France, Roche cuts Balovaptan and Basmisanil; Stanford continue repurposing Vasopressin for Autism

 Repurposing what already exists – cheap, safe, effective and sometimes colourful

Today’s post is nice and simple.

Yet another sub-type of autism is shown in a clinical trial to respond to the cheap drug bumetanide, this time it is children diagnosed with TSC (tuberous sclerosis complex); TSC is a leading genetic cause of autism often used in research.

In France researchers repurposed Folinoral, a lower dose equivalent of Dr Frye’s, and our reader Roger’s, Leucovorin to treat autism with a positive result.  Folinoral is Calcium Folinate, but the dose was just 5mg twice a day, much less than the dose used in the US research.

The potential off-label uses for Verapamil, the old calcium channel blocker helpful in some autism, continue to grow.

Original purpose:  

Lower blood pressure by blocking L-type calcium channels

Alternative uses:

·        Treating bipolar disorder

·        Treating cluster headaches and some migraine

·        Halting the loss of insulin production in people with diabetes

·        Treating diarrhea-predominant irritable bowel syndrome (IBS-D)

·        Treating aggression/anxiety in some autism

We can now add, as our reader Lisa discovered by chance,

·        Treating chronic sinusitis

Patients with severe chronic rhinosinusitis show improvement with Verapamil treatment

"Recently, we became aware that some of the inflammation in chronic rhinosinusitis (CRS) with nasal polyps is generated by the nasal lining itself, when a particular protein pump (P-glycoprotein) is overexpressed and leads to the hyper-secretion of inflammatory cytokines," said senior author Benjamin S. Bleier, M.D., a sinus surgeon at Mass. Eye and Ear and an assistant professor of otolaryngology at Harvard Medical School. "Verapamil is a first-generation inhibitor that is well-established in blocking P-glycoprotein. In some patients with CRS with nasal polyps, we saw dramatic improvement in their symptom scores."

Roche ditching experimental autism drugs

Basmisanil which targets the alpha 5 sub-unit of GABA_A receptors was originally being developed to improve cognition in Down Syndrome; those clinical trials failed. Now Roche have pulled the plug on the trials to improve cognition in Schizophrenia.

Balovaptan was Roche’s expensive bet on Vasopressin to treat autism, covered in earlier posts; it blocks the activity of the V1a vasopressin receptor.  The Balovaptan phase 3 clinical trials have also been cancelled.

Roche culls key phase 3 drug trials, including a cancer med and an autism 'breakthrough'therapy

Stanford still pursuing Vasopressin for autism

Stanford’s bet on Vasopressin for autism is still ongoing.  They had the much simpler idea of just putting some pharmaceutical-grade vasopressin in a nasal spray and trialling that.

Intranasal delivery of drugs to target the brain appeals to me, as do eye drops.  Your eyes are part of the central nervous system, in the case of your nose it appears that drugs are transported directly to the brain from the nasal cavity along the olfactory and trigeminal nerves. 

Mechanism of intranasal drug delivery directly to the brain

One feature of this blog is a belief that central hormonal dysfunction is a core feature of much autism.  The big problem is that you cannot easily measure hormone levels in the central nervous system (CNS) and you may get quite contradictory results measuring hormone levels in blood samples.

Plasma oxytocin and vasopressin do not predict neuropeptide concentrations in human cerebrospinal fluid.

I was encouraged to see that the Stanford vasopressin researchers measured vasopressin in samples from spinal fluid.  They found that children who went on to be diagnosed with autism has very low levels of vasopressin in their brains early in life. Making it a potential biomarker.

Neonatal CSF vasopressin concentration predicts later medical record diagnoses of autism spectrum disorder

Autism spectrum disorder (ASD) is a brain disorder characterized by social impairments. ASD is currently diagnosed on the basis of behavioral criteria because no robust biomarkers have been identified. However, we recently found that cerebrospinal fluid (CSF) concentration of the “social” neuropeptide arginine vasopressin (AVP) is significantly lower in pediatric ASD cases vs. controls. As an initial step in establishing the direction of causation for this association, we capitalized upon a rare biomaterials collection of newborn CSF samples to conduct a quasi-prospective test of whether this association held before the developmental period when ASD first manifests. CSF samples had been collected in the course of medical care of 0- to 3-mo-old febrile infants (n = 913) and subsequently archived at −70 °C. We identified a subset of CSF samples from individuals later diagnosed with ASD, matched them 1:2 with appropriate controls (n = 33 total), and quantified their AVP and oxytocin (OXT) concentrations. Neonatal CSF AVP concentrations were significantly lower among ASD cases than controls and individually predicted case status, with highest precision when cases with comorbid attention-deficit/hyperactivity disorder were removed from the analysis. The associations were specific to AVP, as ASD cases and controls did not differ in neonatal CSF concentrations of the structurally related neuropeptide, OXT. These preliminary findings suggest that a neurochemical marker of ASD may be present very early in life, and if replicated in a larger, prospective study, this approach could transform how ASD is detected, both in behaviorally symptomatic children, and in infants at risk for developing it.

  

Easy to read version: -

Potential autism biomarker found in babies

Cerebrospinal fluid levels of a hormone called vasopressin were lower in babies who went on to develop autism than in those who did not, a study found. 

Cerebrospinal Fluid Vasopressin and Symptom Severity in Children with Autism

 

Cerebrospinal fluid (CSF) arginine vasopressin (AVP) concentration differs between children with and without autism (AUT), predicts AUT diagnosis, and predicts symptom severity. (A) CSF AVP concentration is lower in children with AUT (n = 36) compared to control children (n = 36), whereas (B) CSF oxytocin (OXT) concentration does not differ between groups. 

(C) The effect of CSF AVP concentration on predicted (line) and observed (symbols) group is plotted, corrected for the other variables in the analysis. Children with AUT plotted above, and control children plotted beneath, the dashed line (which represents 50% probability) are correctly classified. Specifically, across the range of observed CSF AVP concentrations, the likelihood of AUT increased over 1,000-fold, corresponding to nearly a 500-fold increase in risk with each 10-fold decrease in CSF AVP concentration (range odds ratio = 1,080, unit odds ratio = 494, β1 ± SE = −6.202 ± 1.898). (D) CSF AVP concentration predicts Autism Diagnostic Observation Schedule (ADOS)–Calibrated Severity Score (CSS) in male but not in female children with AUT.

I think many hormones are likely disturbed in autism and that modifying them is one potential method of treating autism.

At Stanford they have already had success by squirting vasopressin up kids’ noses:-

Hormone reduces social impairment in kids with autism

In a Stanford study of 30 children with autism, intranasal vasopressin improved social skills more than a placebo, suggesting that the hormone may treat core features of the disorder.

A RANDOMIZED CONTROLLED TRIAL OF INTRANASAL VASOPRESSIN TREATMENT FOR SOCIAL DEFICITS IN CHILDREN WITH AUTISM

Stanford University, Department of Comparative Medicine, Stanford Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social impairments and restricted, repetitive behaviors. Despite ASD’s prevalence, there are currently no medications that effectively treat its core features. Accumulating preclinical research suggests that arginine vasopressin (AVP), a neuropeptide involved in mammalian social functioning, may be a possible treatment for ASD. Objective: The goal of this investigation is to examine the safety and efficacy of AVP in the treatment of social deficits in children with ASD. Material and Methods: Using a double-blind, randomized, placebo-controlled, parallel design, we tested the efficacy and tolerability of 4-week intranasal AVP treatment in a sample of N=30 children with ASD aged 6-12 years. Results: AVP compared to Placebo treatment significantly enhanced social abilities in children with ASD as measured by change from baseline in the trial’s primary outcome measure, the Social Responsiveness Scale (a parent-report measure). AVP-related social improvements were likewise evident on clinician impression and child performance-based measures. AVP treatment also diminished anxiety symptoms and some restricted/repetitive behaviors. An endogenous blood AVP concentration by treatment group interaction was also observed, such that participants with the highest pre-treatment blood AVP concentrations benefitted the most from AVP (but not Placebo) treatment. AVP was well tolerated with minimal side-effects. No AVP-treated participant dropped out of the trial, and there were no differences in adverse event rates reported between the AVP and Placebo groups. Finally, no significant changes from baseline were observed in electrocardiogram, vital signs, height and weight, or clinical chemistry measurements after 4-week AVP treatment. Conclusions: These findings suggest that intranasally administered AVP is a well-tolerated and promising medication for the treatment of social impairments in children with ASD.

Using a double-blind, randomized, placebo-controlled, parallel clinical trial design, we found that the 4-week intranasal AVP treatment enhanced social abilities in children with ASD as assessed by the trial’s primary outcome measure, the SRS-2 T score. The robustness of this parent-reported social improvement score was corroborated by convergent evidence from clinician evaluation of the social communication abilities of trial participants and by performance of trial participants on laboratory tests of social cognition. These preliminary findings suggest that intranasally administered AVP may be a promising medication for treatment of core social impairments in children with ASD.

We also sought to investigate whether pretreatment neuropeptide concentrations in blood could predict AVP treatment response. We found that participants with the highest pretreatment AVP concentrations in blood benefitted the most from intranasal AVP treatment. This finding may seem counterintuitive, particularly in light of our recent studies showing that low AVP concentrations in CSF could be used to differentiate ASD cases from non-ASD control individuals (13, 14). One might therefore expect that it would be those children with the lowest endogenous AVP concentrations that stood to benefit the most from intranasal AVP treatment. However, being mindful of safety in this pediatric population, our pilot study used a conservative dose escalation regimen in which children were treated with fairly low doses of AVP throughout much of the trial. Assuming that blood AVP concentrations are related, in some manner, to brain AVP activity—a notion about which there is debate (14, 22–25)—it is possible that participants with lower endogenous AVP concentrations at the trial’s outset were “underdosed” in terms of drug amount or duration of treatment and, therefore, would not benefit as fully from AVP administration as those with higher endogenous AVP concentrations. This interpretation is consistent with our finding that AVP treatment enhanced simple social perceptual abilities independent of pretreatment AVP concentrations in blood, whereas it was only those AVP-treated individuals with higher pretreatment blood AVP concentrations who showed gains in complex social behaviors and a reduction in repetitive behaviors.

Pharmacological intervention

Commercially available injectable sterile AVP was used in this study. It was initially purchased from JHP Pharmaceuticals (Rochester, MI), which was subsequently acquired by Par Sterile Products (Chestnut Ridge, NY) in 2014. The placebo solution was prepared by Koshland Pharm (San Francisco, CA) and consisted of ingredients used in the active solution except for the AVP compound. A pharmacist transferred 25 ml of AVP (20 International Units (IU)/ml) or placebo solutions into standard sterile amber glass bottles with metered (0.1 ml per puff) nasal spray applicators to ensure that the AVP and placebo applicators were visually indistinguishable to the research team. These applicators were coded and given to the Stanford Health Care’s Investigational Drug Service for refrigerated storage (2°C to 8°C) and subsequent dispensing. After the first AVP dose (see below), the dose-escalation regimen at home for all participants involved administration of 4 IU twice daily (or BID) of AVP during week 1 and 8 IU BID of AVP during week 2. Participants aged 6 to 9.5 years then received 12 IU BID of AVP during weeks 3 and 4, whereas participants aged 9.6 to 12.9 years received 16 IU BID of AVP during weeks 3 and 4. A range of possible AVP doses was identified by review of the published literature; the final study doses were then determined in close consultation with the FDA.

A few years ago I did write about the hormone TRH as a potential means of improving autism.  TRH can also be squirted up your nose, although I favoured an oral TRH super-agonist called Taltirelin/Ceredist.

I also suggested that DHED, an orally active, centrally selective prodrug of estradiol, could well be a therapeutic in autism. DHED should give all the benefits of the female hormone estradiol, without any side-effects outside the CNS.  Many of the benefits are via ROR alpha.

Without having samples of spinal fluid, identifying, let alone treating, central hormonal dysfunction is rather a matter of guesswork.

Hormones are very much interrelated and perform different functions in different parts of the body, so it would be easy to get unwanted effects, as with estradiol, if taken orally.

  

Bumetanide for TSC (Tuberous Sclerosis Complex)

A small trial in children with TSC (Tuberous sclerosis complex) has shown that bumetanide improved their features of autism (social behavior, irritability and hyperactivity) but did not reduce seizures.

Effects of bumetanide on neurodevelopmental impairments in patients with tuberous sclerosis complex: an open-label pilot study

Conclusion

This pilot study indicates the potential efficacy of bumetanide on behavioral problems in young patients with TSC. Bumetanide improved irritable, explosive, and social behavior in the majority of patients in this sample and treatment was well tolerated.

Folinic Acid for Autism, but at a lower dose than Dr Frye

I did recently complete my trial of generic Calcium Folinate at something like Dr Frye’s Leucovorin dose.

I found that it did indeed have a positive effect on the use of expressive language.  It prompted the use of more complex sentences.

The downside was that it did also cause aggressive/violent outbursts, so I put it in my “rejected” pile of therapies.  

I was interested to see that in France a trial has been carried out using a lower dose than that proposed by Dr Frye.  Is it possible to get benefits without the side effects? 

Folinic acid improves the score of Autism in the EFFET placebo-controlled randomized trial  

Highlights 

Folinic acid treatment is well tolerated in children with Autism spectrum disorders.

Folinic acid treatment shows improvement in Autism Diagnostic Observation Schedule score.

Effect of 10 mg/d folinic acid should be confirmed by a larger a multi-center trial.

Autism spectrum disorders (ASD) are influenced by interacting maternal and environmental risk factors. High-dose folinic acid has shown improvement in verbal communication in ASD children. The EFFET randomized placebo-controlled trial (NCT02551380) aimed to evaluate the efficacy of folinic acid (FOLINORAL®) at a lower dose of 5 mg twice daily.

Nineteen children were included in the EFFET trial. The primary efficacy outcome was improvement of Autism Diagnostic Observation Schedule (ADOS) score. The secondary outcomes were the improvement in ADOS sub scores communication, social interactions, Social Responsiveness Score (SRS) and treatment safety.

The global ADOS score and social interaction and communication sub scores were significantly improved at week 12 compared to baseline in the folinic acid group (P = 0.003, P = 0.004 and P = 0.022, respectively), but not in the placebo group (P = 0.574, P = 0.780, P = 0.269, respectively). We observed a greater change of ADOS global score (−2.78 vs. −0.4 points) and (−1.78 vs. 0.20 points) in the folinic acid group, compared to the placebo group. No serious adverse events were observed.

This pilot study showed significant efficacy of folinic acid with an oral formulation that is readily available. It opens a perspective of therapeutic intervention with folinic acid but needs to be confirmed by a multi-center trial on a larger number of children.

  

Covid-19

There was concern that people with severe autism might be at increased risk during the current pandemic and indeed the death rate among people with intellectual disability/learning disability/mental retardation did double from 240 a month to 480 a month in the UK.  The real scandal though was deaths in care homes for the elderly, in countries with advanced healthcare systems, where tens of thousands of extra deaths have occurred.

In “advanced” healthcare systems like the UK, early in the epidemic, elderly people caught Covid-19 in hospital and when they returned to their care home, they infected others.  Care workers who are allowed/forced to work in multiple care homes then caught the virus in one home and transmitted it to the others.  Nobody was tested until care homes had already become breeding grounds for the virus.

In Hong Kong they report zero covid-19 deaths in care homes.  Elderly people could not return to their care home from hospital without testing negative for the virus, and procedures were in place to release elderly patients from hospital first to repurposed hotels, where they stayed until negative for the virus. Due to their grim experience with the 2003 SARS epidemic, Hong Kong already had very strict measures in place to limit infections and they even had regular rehearsals in care homes of the procedures to implement in future pandemics.

Where we live there was an outbreak in a care home and the authorities’ reaction was to arrest the boss of the care home.  I suppose that is one way to get other care homes to take matters seriously. We even had soldiers posted outside care homes to stop people entering.  In New York, Cuomo’s threat to care homes was that you might eventually lose your license to operate if you flout the rules. If most care homes are flouting the rules, they cannot all lose their licenses.

Some rich Western countries apparently implemented their much-vaunted flu pandemic procedures.  It looks like they have much to learn from other places, from Hong Kong to Greece, who did very much better.  Greece implemented a draconian lock down, very early, and has had a tiny number of cases and just 166 deaths. When Greece re-opens in July to tourists from high risk countries (UK, France, Italy, Spain etc) we will see what happens.

I do wonder why so many people are living in care homes. In Sweden, I saw on TV, one lady complaining that her fit and healthy father, capable of walking a few miles/km had caught covid-19 in his care home, was refused transfer to hospital and later died.  Why was he sent to live a care home in the first place?

Milan has an old care home called Pio Albergio Trivulzia ("Baggina"), it had over a thousand residents and media reports 200+ covid deaths.

There are horrific cases in the UK of young adults being sent to live in small mental hospitals by their parents; they subsequently deteriorate and some have even died.  Why did the parents hand their children over in the first place?  They thought they could not cope at home, but clearly some dedicated institutions have even less capacity to care. 

Conclusion

Re-purposing existing cheap drugs to treat a different medical condition makes a lot of sense, but it is not going to make the inventor or the drug firm much money.  It is not popular with drug producers.

Developing new drugs to treat any neurological condition looks great in the early stages of research and then they all seem to fade way, wasting many tens of millions of dollars.  Don’t raise your hopes.

Is intranasal vasopressin the smartest hormone to choose to modify?  It is possible today, using existing products and appears to be safe, which are the most important issues. I think there is more potential beyond this single hormone.

Treat autism and intellectual disability/mental retardation medically, so those people can live more normally, be more fulfilled and do not later need such expensive care home provision. It is a win-win strategy.

Cohesinopathies, including Cornelia de Lange syndrome and the overlapping phenotype SMC3, causing altered gene expression

Today’s post is in response to a request from a parent interested in a very rare genetic condition involving the SMC3 (Structural Maintenance of Chromosomes protein 3) gene, it is  cohesinopathy related to  Cornelia de Lange syndrome, but really better considered a distinct condition.  A cohesinopathy is a condition affecting the cohesin complex.  This subject does get very complicated and requires a biology lesson.

Mitosis

Mitosis is the process where one cell produces an identical copy of itself and one becomes two.

In the figure below consider the red chromosome; it has to become two red chromosomes and just before this occurs, you have two identical “chromatids” that are joined together at the “centromere”, like an “H” shape. The chromatids later separate and become two identical red chromosomes in two identical cells. 

In each cell there are two copies (here shown red and blue) of 22 chromosomes.  In addition is the problematic X chromosome, where boys have no second copy, but girls do, giving them a level of protection. Boys have a Y chromosome in place of the second X chromosome.

The cohesin complex is like a hinged claw that holds together sister chromatids, in the “H” shape during mitosis, enabling proper chromosome segregation and avoiding premature separation.  If the separation is improperly executed, this could result in the segregation of both sisters to the same cell thereby generating an abnormal number of chromosomes (aneuploidy).  Aneuploidy is the presence of an abnormal number of chromosomes in a cell; for example a human cell having 45 or 47 chromosomes instead of the usual 46.

In people with Down syndrome, the most common human condition due to aneuploidy, there are three copies of all or part of chromosome 21, rather than the usual 2 copies.  It can be referred to as trisomy 21.  This then results in over expression of up to a few hundred genes and this then appears to us as a person with Down syndrome.

Some researchers consider Down Syndrome a cohesinopathy:-

The cohesin complex and its roles in chromosome biology

Defects in cohesin function have been linked to several human diseases that are therefore also referred to as “cohesinopathies.” The most frequent of these diseases is Down syndrome (trisomy 21), which in the majority of cases is caused by missegregation (“nondisjunction”) of chromosome 21.

Most research lists the following as cohesinopathies:-

·         Cornelia de Lange Syndrome

·         Roberts syndrome

·         Warsaw breakage syndrome

·         Many types of cancers

You have to understand what cohesin is and how it attaches and releases from your DNA.  Imagine a hinged claw opening and grabbing your DNA (the loading process) and later the hinge opens and the claw separates from your DNA (the unloading process). Cohesion is when two pieces of different DNA are joined.

This cohesin claw grabs hold of your DNA and later has to let go.  If any part of that process goes awry, you have a Cohesinopathy.

Several proteins are involved in the cohesion claw and more are involved in the opening and closing process.

The claw is made up two jaws, SMC1 and SMC3 with a hinge at the top.

Our reader’s child has a mutation in the SMC3 gene that encodes the jaw on the right.

Cornelia de Lange Syndrome is the name given if have a mutation of NIPBL, SMC1A, SMC3, RAD21 and HDAC8.

To be more precise, most Cornelia de Lange Syndrome is really a condition where the jaw closing process is disturbed by a lack of the protein NIPBL.  NIPBL has other functions beyond cohesin and this is why people with a NIPBL mutation have more physical differences than people with a SMC3 mutation.

Some people have a mutation in HDAC8 and this disrupts the jaw opening process that release the bite from the DNA.

People with a SMC1A or SMC3 mutation just have a faulty left/right claw.

People with a RAD21 mutation have the little part at the bottom of the claw not properly functioning.

Other functions of SMC3 and snippets from the research:-

Although the SMC3 protein is found primarily in the nucleus, some of this protein is transported out of cells. The exported protein, which is usually called bamacan, may be involved in sticking cells together (cell adhesion) and cell growth. Bamacan is a component of basement membranes, which are thin, sheet-like structures that separate and support cells in many tissues. Little else is known about the function of this protein outside the cell, but it appears to be important for normal development.

Our data indicate that SMC3 and SMC1A mutations (1) contribute to ∼5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.

At least 15 mutations in the SMC3 gene have been found to cause Cornelia de Lange syndrome, a developmental disorder that affects many parts of the body. Researchers estimate that mutations in this gene account for 1 to 2 percent of all cases of this condition.

Most of the SMC3 gene mutations that cause Cornelia de Lange syndrome either change single protein building blocks (amino acids) in the SMC3 protein or add or delete a small number of amino acids in the protein. Each of these mutations alters the structure and function of the protein, which likely interferes with the activity of the cohesin complex and impairs its ability to regulate genes that are critical for normal development. Although researchers do not fully understand how these changes cause Cornelia de Lange syndrome, they suspect that altered gene regulation probably underlies many of the developmental problems characteristic of the condition.

Studies suggest that mutations in the SMC3 gene tend to cause a form of Cornelia de Lange syndrome with relatively mild features. Compared to mutations in the NIPBL gene, which are the most common known cause of the disorder, SMC3 gene mutations often cause less significant delays in development and growth and are less likely to cause major birth defects

The cohesin complex and its roles in chromosome biology

Cohesinopathies Defects in cohesin function have been linked to several human diseases that are therefore also referred to as “cohesinopathies.” The most frequent of these diseases is Down syndrome (trisomy 21), which in the majority of cases is caused by missegregation (“nondisjunction”) of chromosome 21 during meiosis I in oocytes (Hassold and Hunt 2001; Gilliland and Hawley 2005). Other trisomies are thought to occur with similar frequencies during meiosis, but with the extremely rare exceptions of trisomies 13 and 18, these situations are lethal during embryogenesis. As women age, the frequency with which nondisjunctions occur in oocytes increases dramatically.  This maternal age effect correlates with an increased frequency of unpaired (univalent) chromosomes and precociously separated sister chromatids that can be observed in oocytes, suggesting that defects in sister chromatid cohesion may be one of the major causes of nondisjunction (Angell 1995; Wolstenholme and Angell 2000; Pellestor et al. 2003, 2006). Experimental support for this hypothesis comes from the observation that mice in which the meiosis-specific Smc1 gene has been mutated show age-related defects in oocytes that mimic the situation observed in humans (Hodges et al. 2005). It is therefore possible that defects in cohesin or cohesin regulators are a major cause of Down syndrome. How these defects may occur, and why they would be agerelated, is unclear. However, it is interesting to note that in budding yeast cohesion can normally only be established during DNA replication (see above). If the same were true for mammalian oocytes, where cohesion is established during premeiotic DNA replication during prenatal development, cohesion would have to be able to persist for many years or even decades. Whether cohesin subunits can be dynamically exchanged during this long period (for example, by Scc2/Scc4 and Wapl-mediated mechanisms), or whether cohesion can be established de novo (as is the case in yeast under certain conditions), remains unknown. In either case, it is conceivable that cohesion slowly deteriorates over time and that the frequency of nondisjunction events therefore increases with age. The high frequency with which aneuploidy can be observed in human oocytes and the age dependency of this phenomenon imply that mutations in cohesin genes are not or are only rarely responsible for nondisjunction events. However, Cornelia de Lange and Roberts/SC phocomelia syndrome have been linked recently to hypomorphic mutations in genes of cohesin subunits or regulators. Cornelia de Lange syndrome is characterized by growth defects, various developmental abnormalities, and mental retardation (for review, see Dorsett and Krantz 2008). About half of all investigated cases are caused by loss-of-function mutations in one allele of the NIPBL gene, the human ortholog of Scc2 (Krantz et al. 2004; Tonkin et al. 2004), and a few cases have been identified in which the genes encoding Smc1 and Smc3 have been mutated (Musio et al. 2006; Deardorff et al. 2007). Because sister chromatid cohesion is largely normal in cells derived from these patients, a defect in another cohesin function such as gene regulation may be the cause of the disease. Roberts/SC phocomelia syndrome is also characterized by developmental defects, although the syndrome is clinically distinct from Cornelia de Lange syndrome (for review, see Dorsett 2007). Roberts/SC phocomelia syndrome has been linked to mutations in Esco2 (Schule et al. 2005; Vega et al. 2005), and consistent with the suspected role of this enzyme in cohesion establishment, defects in centromeric cohesion have been observed in cells derived from Roberts syndrome patients (German 1979; Tomkins et al. 1979). Whether these cohesion defects are the direct cause of the disease, or if defects in possible other functions of Esco2 are more relevant, is still unknown. O

Enigmatic Ladies of the Rings: How Cohesin Dysfunction Affects Myeloid Neoplasms Insurgence

“One of the biological meaning of a living organism is the possibility to divide by replicating DNA and generate a new organism. To accomplish this, the genome duplication should be error free and the daughter cell should properly inherit the genetic material from the mother cell. The cohesin proteins are required during this multistep process: in interphase to maintain genome stability during DNA double strand break repair, in S-phase to enforce Sister Chromatid Cohesion (SCC) throughout DNA replication, and in M-phase to ensure proper chromosome distribution into dividing cells. Since the cohesin protein complex has essential roles in the cell, members of the cohesin complex are found from bacteria to humans and are evolutionary and functionally conserved. The ability of cohesin to perform these functions resides in their property to encircle the DNA, creating topological links between chromatin fibers. To mediate sister chromatids tethering and segregation or DNA double strand break repair, the cohesin complex binds to the DNA in a trans conformation. However, cohesin might also encircle the DNA in cis, forming chromatin loops and contributing to gene regulation by modulating genome architecture or joining two distant segments of the genome. In vertebrates, the ring that embraces the DNA is formed by coiled-coil heterodimers of Structural Maintenance of Chromosomes (SMC) subunits SMC1 and SMC3, by the alpha-kleisin subunit RAD21 that brings in connection the ATPase head domains of SMC proteins and stabilizes their interactions, and by the stromal antigens STAG1/STAG2 (SA1/SA2). Although cohesin proteins are intrinsically able to topologically bind to the DNA, the loading of the complex is not efficient in the absence of the NIPBL/MAU2 heterodimer. As stated by their name, cohesin becomes “cohesive” only when the SMC3 head domain subunits are acetylated by the acetyl-transferase ESCO1/ESCO2. The release of the complex from the DNA is achieved by the separase-mediated proteolytic cleavage of RAD21, the HDAC8-mediated de-acetylation of SMC3, or the opening of the RAD21-SMC3 complex controlled by accessory proteins such as CDCA5 (soronin), PDS5 and WAPL.”

Mutations in Cohesin Complex Members SMC3 and SMC1A Cause a Mild Variant of Cornelia de Lange Syndrome with Predominant Mental Retardation

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to ∼5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.

The cohesin proteins compose an evolutionarily conserved complex whose fundamental role in chromosome cohesion and coordinated segregation of sister chromatids has been well characterized across species.^1,2 Recently, regulators of cohesin and a structural component of the complex have surprisingly been found to cause phenotypically specific human developmental disorders when mutated. Mutations in NIPBL, the vertebrate homolog of the yeast Sister chromatid cohesion 2 (Scc2) protein, a regulator of cohesin loading and unloading, are responsible for ∼50% of cases of Cornelia de Lange syndrome (CdLS [MIM #122470 and #300590]).^3–5 Mutations in another cohesin regulator, ESCO2, have been found to result in Roberts syndrome and SC phocomelia.^6,7 Two mutations in the cohesin structural component SMC1A (for structural maintenance of chromosomes 1A based on revised ^HUGO nomenclature; also called “SMC1L1”) were recently found to result in an X-linked form of CdLS.⁸ The conserved developmental perturbations seen in these disorders are likely the result of disruption of the cohesin complex’s role in facilitating long-range enhancer promoter interactions and subsequent transcriptional dysregulation.^9,10

CdLS is a dominantly inherited genetic multisystem developmental disorder. The clinical features consist of craniofacial dysmorphia, hirsutism, malformations of the upper extremities, gastroesophageal dysfunction, growth retardation, neurodevelopmental delay, and other structural anomalies (see facies and limbs of patient 1P in fig. 1). The mental retardation seen in CdLS, although typically moderate to severe, displays a wide range of variability.

Facies and hands of classic CdLS in SMC3- and SMC1A-mutation–positive individuals. Proband numbers are indicated. A “P” following the number indicates proband, and an “S” indicates an affected sister. Facial features and upper extremities are shown for 1P, a patient with classic CdLS and a truncating NIPBL mutation; 2P, a male with a sporadic SMC3 E488del mutation; 3P, a female with a sporadic V58-R62del mutation; 4P, a male with a sporadic F133V mutation; 6P, a male with a sporadic R496C mutation; 7P and 7S, two sisters of family 2 with the R496H mutation and mosaicism in the unaffected parent; 8P and 8S, two sisters of family 1 who share a R496H mutation; 9P, a female with a sporadic R496H mutation; 10P, a male with a sporadic R711W mutation; 11P, a female with a sporadic R790Q mutation; and 12P, a female with a sporadic F1122L mutation.

Here is a graphical summary of the key Cohesinopathies and where they result from:-

Source: Genetic basis of cohesinopathies

The other role of SMC3

In addition to forming part of the cohesin chromosome “claw”, it also forms a complex with SMC1 called RC-1 which is involved in DNA repair.

Over expression of SMC3 is thought to act via cohesin and RC-1 to play a role in cancer formation and growth.  This is why some malignancies are classed as cohesinopathies.

In the people with SMC3 mutation, classed as part of Cornelia de Lange syndrome, there appears to be under-expression of SMC3.

SMC3 has a third unrelated function where the protein tends to be called by the alternative name bamacan.  This time SMC3/bacman has completely different functions that are poorly documented, but are unrelated to cohesion or RC-1.  Bamacan is an abundant basement membrane protein.

It looks like a lack of SMC3 has a knock-on effect of SMC1.

Recall that the cohesin claw is made up of SMC3 on one side and SMC1 on the other.

Imbalance of SMC1and SMC3 Cohesins Causes Specific and Distinct Effects

  

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

How to treat an SMC3 mutation?

“altered gene regulation probably underlies many of the developmental problems characteristic of the condition” … Okay, but which genes are affected 

Start by looking at the public data on which proteins that interact with SMC3

Interacting Proteins

https://www.genecards.org/cgi-bin/carddisp.pl?gene=SMC3

The 5 most significant:-

Not surprisingly SMC3 most important interactions are with other proteins/genes that are involved with directly with cohesin.

PDS5B associates with WAPL to stimulate the release of cohesin from DNA but during DNA replication PDS5 promotes acetylation of SMC3 by ESCO1 and ESCO2.

The exception is CDC5L (Cell division cycle 5-like protein), it is DNA-binding protein involved in cell cycle control, it may act as a transcription activator.

Look a bit deeper.  Consider the 25 most significant interactions with SMC3:-

When we look at the top 25 protein interactions, we see other cohesion-related proteins appearing like NIPBL (the core Cornelia de Lange syndrome protein), plus STAG1, STAG2 etc.

Notably at the top of the chart we see 4 proteins from the BCL2 family.

Regular readers may recall that BCL2 are known to be both autism and cancer genes.

Bcl-2 (B-cell lymphoma 2), encoded in humans by the BCL2 gene, is the founding member of the Bcl-2 family of regulator proteins that regulate cell death, by either inhibiting or inducing apoptosis.

Apoptosis plays an important role in regulating a variety of diseases. For example, schizophrenia is a psychiatric disorder in which an abnormal ratio of pro- and anti-apoptotic factors may contribute towards pathogenesis.

SMC3 mutations

SMC3 mutations are reported to be associated with microcephaly (small heads).

We can likely put this in the hypo-active pro-growth signaling pathways category.

Source:  Characterizing Autism Spectrum Disorders by Key Biochemical Pathways

If we look directly at the signalling pathways associated with SMC3, there is a rather bewildering number, but read on.

Many pathways relate to copying/repairing DNA, making proteins and making new cells.

Pathways and interactions for SMC3

1.     Integrin Pathway

2.     ERK Signaling

3.     Cell Cycle, Mitotic

4.     Mitotic Metaphase and Anaphase

5.     SUMOylation

6.     Phospholipase-C Pathway

7.     Mitotic Telophase/Cytokinesis

8.     CDK-mediated phosphorylation and removal of Cdc6

9.     Meiosis

10.Metabolism of proteins

11.Mitotic Prometaphase

12.Chromatin Regulation / Acetylation

13.DNA Damage

14.Cell cycle

15.Oocyte meiosis

16.ATM Pathway

17.Cell cycle_Spindle assembly and chromosome separation

18.MECP2 and Associated Rett Syndrome

19.Retinoblastoma (RB) in Cancer

If we look at the ERK signalling pathway , we see that one important element is RAS.  In earlier posts we came across the term RASopathy.  These conditions tend to be associated with Intellectual Disability (ID)/ Mental Retardation (MR).  This is a feature of SMC3 and a variable feature of Cornelia de Lange syndrome.

Also note PAK.

RAS, BCL-2, PTEN. Wnt signalling are all interrelated.

I did write extensively about PAK inhibitors (FRAX 486, Ivermectin etc).

You can have too much or too little RAS.

Based on previous posts we know that BCL2 will affect Wnt signaling and this may well lead to the thinned out dendritic spines.

We saw in an old post that at MIT they are suggesting that errors in synaptic protein synthesis are involved in several types of autism and that these errors can potentially be corrected using either positive or negative stimulators of the receptor mGluR5.

So for SMC3 we should need a PAM (positive allosteric modulator) of mGluR5

Ketamine, Memantine, D-Cycloserin, Magnesium, Fenobam and yet more as Glutamatergic Modulators in Autism (and Fragile X)

We can also target Wnt signaling as a means to try and improve dendritic spine morphology.  We just have to get things the right way around. We can enhance Wnt/β-Catenin Signaling with Simvastatin or indeed lithium.

Abnormal hair development is a feature of SMC3 and that would fit with abnormal Wnt signalling and abnormal dendritic spines.

As I have pointed out in an earlier post the BCL-2 protein is also involved in hair growth and greying.

NIPBL-type Cornelia de Lange syndrome

I did suggest that it is not helpful to call SMC3 a sub-type of Cornelia de Lange syndrome, because the conditions are not the same.

Just as SMC3 has multiple functions so does NIPBL.

These function do overlap in the case of cohesin, but it looks like under-expression of NIPBL has more wide ranging consequences than under-expression of SMC3, in particular when it comes to physical features.  This makes sense because of the roles SMC3 and NIPBL have beyond cohesin. 

In the case of lack of the protein NIPBL, you get genuine Cornelia de Lange syndrome.  This specific condition looks to have a possible therapy using a PKR inhibitor, but this will not work for SMC3.  I include it here in case anyone interested in NIPBL reads this post.


NIPBL Controls RNA Biogenesis to Prevent Activation of the Stress Kinase PKR

NIPBL, a cohesin loader, has been implicated in transcriptional control and genome organization. Mutations in NIPBL, cohesin, and its deacetylase HDAC8 result in Cornelia de Lange syndrome. We report activation of the RNA-sensing kinase PKR in human lymphoblastoid cell lines carrying NIPBL or HDAC8 mutations, but not SMC1A or SMC3 mutations. PKR activation can be triggered by unmodified RNAs. Gene expression profiles in NIPBL-deficient lymphoblastoid cells and mouse embryonic stem cells reveal lower expression of genes involved in RNA processing and modification. NIPBL mutant lymphoblastoid cells show reduced proliferation and protein synthesis with increased apoptosis, all of which are partially reversed by a PKR inhibitor. Non-coding RNAs from an NIPBL mutant line had less m⁶A modification and activated PKR activity in vitro. This study provides insight into the molecular pathology of Cornelia de Lange syndrome by establishing a relationship between NIPBL and HDAC8 mutations and PKR activation.

 

NIPBL and cohesin may contribute to gene expression in different ways. For instance, NIPBL may be involved in the maintenance of NFRs, while cohesin may be important in long-distance interactions. Due to these different molecular functions, loss of function may not have equivalent effects on gene expression. For example, the gene expression profiles of cells upon NIPBL or cohesin knockdown are different (Muto et al., 2011; Zuin et al., 2014). Our study further supports this idea since CdLS LCLs with mutations in SMC1A or SMC3 do not show PKR activation. A previous study showed that NIPBL directly interacts with histone-deacetylating enzymes HDAC1 and HDAC3 in human cells (Jahnke et al., 2008), suggesting that NIPBL may initiate the chromatin-remodeling processes through the recruitment of these HDACs in transcriptional regulation. The budding yeast ortholog of NIPBL, SCC2, may participate in transcriptional regulation by maintaining NFRs through the association with remodels the structure of chromatin (RSC; Lopez-Serra et al., 2014). In the future, it will be important to continue to dissect the molecular role of NIPBL and cohesin in gene expression, since this knowledge will help us understand how loss of function leads to human disease.

In summary, we suggest that NIPBL facilitates a gene expression program compatible with normal RNA biogenesis. Upon NIPBL loss of function, there is reduced expression of RNA-processing genes, which correlates with the generation of unmodified RNAs, including m⁶A deficiency. Such aberrant ncRNAs could activate the PKR-signaling cascade, leading to poor cell proliferation, protein synthesis, and apoptosis. Importantly, treatment with a PKR inhibitor can partially rescue these defects. The findings shed light on the molecular etiology of CdLS by highlighting the activation of PKR in the NIPBL and HDAC8 mutant cells. Identification of elevated PKR activity suggests a new avenue for disease management, namely the use of PKR inhibitors to ameliorate cellular stress associated with CdLS.

 

 

NIPBL and cohesin may contribute to gene expression in different ways. For instance, NIPBL may be involved in the maintenance of NFRs, while cohesin may be important in long-distance interactions. Due to these different molecular functions, loss of function may not have equivalent effects on gene expression. For example, the gene expression profiles of cells upon NIPBL or cohesin knockdown are different (Muto et al., 2011; Zuin et al., 2014). Our study further supports this idea since CdLS LCLs with mutations in SMC1A or SMC3 do not show PKR activation. A previous study showed that NIPBL directly interacts with histone-deacetylating enzymes HDAC1 and HDAC3 in human cells (Jahnke et al., 2008), suggesting that NIPBL may initiate the chromatin-remodeling processes through the recruitment of these HDACs in transcriptional regulation. The budding yeast ortholog of NIPBL, SCC2, may participate in transcriptional regulation by maintaining NFRs through the association with remodels the structure of chromatin (RSC; Lopez-Serra et al., 2014). In the future, it will be important to continue to dissect the molecular role of NIPBL and cohesin in gene expression, since this knowledge will help us understand how loss of function leads to human disease.

In summary, we suggest that NIPBL facilitates a gene expression program compatible with normal RNA biogenesis. Upon NIPBL loss of function, there is reduced expression of RNA-processing genes, which correlates with the generation of unmodified RNAs, including m⁶A deficiency. Such aberrant ncRNAs could activate the PKR-signaling cascade, leading to poor cell proliferation, protein synthesis, and apoptosis. Importantly, treatment with a PKR inhibitor can partially rescue these defects. The findings shed light on the molecular etiology of CdLS by highlighting the activation of PKR in the NIPBL and HDAC8 mutant cells. Identification of elevated PKR activity suggests a new avenue for disease management, namely the use of PKR inhibitors to ameliorate cellular stress associated with CdLS.

 

Conclusion

Our reader with a child with an SMC3 mutation knows what the route problem is, but there is no easy way to modify the expression of the SMC3 gene.

Since quite a lot is known about what SMC3 does and we know it is a microcephaly type of autism with ID/MR, there is quite a long list of things that may be partially effective.

You cannot really change physical variations, but we have seen that you can fine tune brain function.

As we have seen, it is very easy to be too clever and actually get things exactly the wrong way round for example with agonists and antagonists working in reverse.

Also, the clever MIT researchers have all ultimately failed to develop new drugs that work for single gene types of autism. 

I would be more governed by what safe therapies exist than can be repurposed.  That list really is not very long.  Then I would start with that and see what might potentially be helpful in this specific case.

For example, is SMC3 going to be bumetanide responsive? A very wide range of disorders from Down Syndrome, Fragile X, to Parkinson’s are reported to benefit. It is impossible to know for sure if it will work, but it is oh so simple to find out.  Just make a trial.

Is the PAK inhibitor FRAX 486 going to help? Maybe, but you cannot easily get it, so best not to think too much about it.

I would definitely look at the statins, I would try Simvastatin, Lovastatin and Atorvastatin.  Why?  They are cheap, safe and available and each one has slightly different effects that might well help.  In the case of SMC3, we know BCL2 is involved and we know that statins can modulate this.

Lithium might be helpful

Regarding mGluR5, the options are rather limited.  It is much easier to damp it down than to increase its effect.  The Fragile X research failed. 

I would also try the usual popular interventions, such as

·        NAC

·        Sulforaphane (our reader tpes is a big fan of the French product Prostaphanae)

·        Dr Frye’s Leucoverin might have an effect, a recent successful trial in France uses a much lower dose than Dr Frye (5mg twice a day of calcium folinate) 

If an MRI was carried out, you could look at myelination.  It might be reduced; if it was then we have a long list of pro-myelination therapies (Clemastine, Ibudilast ...).  You could try the antihistamine Clemastine for a couple of months and skip the MRI.

I thought that both IGF-1 and Baclofen might be worth considering. Insulin-like growth factor 1 (IGF-1) is produced in response to stimulation by growth hormone GH. One effect of Baclofen is to increase GH and so also IGF-1. Both Baclofen and IGF-1 affect BCL-2. Both IGF-1 and (ar)baclofen have been trialed in autism. Baclofen is a cheap tablet, but IGF-1 needs a subcutaneous injection.  Intranasal insulin might be a way to affect IGF-1 receptors in the brain. In microcephaly we can assume growth hormones/factors have been disturbed. 

If there are GI symptoms, these can be treated by numerous autism/GI therapies and will improve function.

Looking at inflammatory cytokines might show up some anomalies and this blog has documented numerous therapies.  For example, Pentoxifylline is cheap generic drug that Maja recently commented about having success with, but there are very many others.  

I think to treat an SMC3 mutation you would need a helpful and enlightened doctor, who understands risk, in particular the risk of doing nothing.