Originally posted 11 December 2021 Updated 14 June 2022
Multiple studies of Phelan-McDermid syndrome (PMS) have demonstrated worse intellectual outcomes with larger chromosome deletions. Genetic studies on large populations of people with intellectual disabilities show that the measure called “pLI” is useful for predicting IQ as a function of deletion size (see PMS, IQ and why interstitial deletions matter). The high pLI PMS genes are the most import genes of 22q13.3, the chromosome site of PMS problems (see Which PMS genes are most important?). In the past I have written about which PMS genes might cause the structural abnormalities in brain MRIs of PMS patients (see Which genes cause brain abnormalities in Phelan McDermid syndrome?). But, just this past year, scientists have discovered that a high pLI PMS gene is vital to the proper microstructure of the brain. Based on a series of very careful studies in mouse embryos, this group of researchers uncovered the special role of PMS gene, Phf21b. The high pLI of Phf21b (0.987 out of a possible 1.00) already tells us that it is an important PMS gene. The scientific team now tells us why it is so important in their 20+ page scientific paper (Phf21b imprints the spatiotemporal epigenetic switch essential for neural stem cell differentiation.) They solved the mystery of Phf21b and the whole PMS community benefits from their work. The paper is difficult reading, but open to the public.
To understand the new research we need to take a moment to review how our brains develop in the uterus. PMS is a neurodevelopmental disorder. That is, it is a disorder of brain development. Our brains assemble into complex structures with even more complex synaptic connections as they grow. The higher centers for thought, precise movement and proper interpretation of our senses are located in the cortex. The cortex is the part of the brain that we can see in photographs of the human brain. It is the most developed and expanded part of the brain that humans have.
I have borrowed part of figure 8C from the paper on Phf21b to show the developing cortex in a mouse. It shows that neurons (the specialized brain cells responsible for thinking and acting) arise as general purpose cells from the ventricular zone (VZ, magenta color) and subventricular zone (SVZ, green color). Together, these are called progenitor cells. They are especially good at reproducing at a high rate to fill the areas above the SVZ. The rapidly growing and reproducing cells actually climb their way into final position. I found this video on the Internet that shows the process (Neurogenesis Animation). The rapid growth is shown 2:39 (min:sec) into the animation. This process creates about 100,000 cells per mm3 in the developing mouse. The process is driven by a large group of specialized genes that fuel the rapid growth and reproduction. These genes are collectively called, cell cycle genes. At a certain very crucial moment in the life of a progenitor cell, the cell stops growing and reproducing. The cell switches from being a progenitor to a neuron, and then spends the rest of its life as a neuron. Phf21b is the gene that tells the progenitor cells when to switch.
How does one gene stop the frenetic activity of progenitor cells? How does it switch off the many cell cycle genes involved in rapid growth and reproduction? The scientists spent some time figuring this out. Phf21b protein is not produced much in progenitor cells until the proper moment. Then, Phf21b protein production suddenly increases. It goes into the nucleus and searches out the precise spot in the DNA that regulates each cell cycle gene of the cortex. Phf21b is an epigenetic switch. It wraps around the regulatory site of each growth gene, and assembles a toolkit of proteins to shut that gene off.
Here is the right half of figure 8C from the paper. The magenta neural progenitor (NPC) cell on the left gets transformed into a neuron (blue cell on the right) by the action of Phf21b (green molecule in the middle). Through several steps involving other molecules (Rcor1, Hdac2, and Lsd1), Phf21b acts as a precision switch. It turns off just the cell cycle genes that promote rapid cortical growth. Phf21b literally hits the brakes on growth to trigger the creation of neurons in the cortex of mammals.
The experiments used by the authors to figure out this exquisite mechanism were complicated and, in some cases, exceedingly delicate. The team of nine researchers represented six institutes in three different countries. Among the many experiments undertaken, they showed the impact of disrupting this mechanism. With insufficient Phf21b, the normal pattern of neurons in the cortex is disrupted.
It has recently been shown that improper regulation of proliferation, both too little or too much, is associated with autism.
In spite of the groundbreaking work, certain questions remain. First, what aspects of the PMS disorder (what phenotypic characteristics) are driven by loss of PHF21B in humans? One hint comes from a paper published in 2014 by Disciglio and colleagues. It describes nine patients with interstitial deletions of 22q13. Although these authors argued that they discovered a new syndrome, the phenotype readily overlaps with PMS, with intellectual disability and speech delay being the two central traits. Many people (including myself) see these simply as cases of PMS (see The four types of Phelan McDermid syndrome). Regardless, of the nine patients in the study, eight are missing PHF21B and the exception (called patient #4) had a deletion that was very near PHF21B (within 84 Kb). That patient was arguably the least affected individual, as well. Thus, PHF21B may have been a major driver of the manifestations (symptoms) seen in these patients. More cases of PMS with interstitial deletions (PMS type 2) are needed to confirm this observation. What would be especially useful would be to find patients with pathological variants of PHF21B. As more genetic testing is done in newborns, especially whole exome or whole genome sequencing, there will be more cases. Why should we expect a growth in cases? We already know the two critical characteristics: 1) loss of one functional copy of PHF21B is pathological (the pLI is greater than 0.9) and 2) loss of one functional copy of PHF21B is compatible with life (there are patients with PMS type 1 that have deletions that include PHF21B because their deletions are greater than 5.81 Mb). About 40% of all identified individuals with PMS are missing this gene (see Understanding deletion size). So, we know these patients are common in PMS.
The second unanswered question is what, exactly, does the loss of PHF21B do to the human cortex once development is complete. The human primate cortex is far more complex than the rodent cortex (see Mashiko et al 2012, Chen et al 2016, Preuss and Wise 2022). Still, the mouse cortex can provide insights into the dysplasia likely to occur in humans. One research direction would be to compare the histology of rodent cortex in a Phf21b knockout mouse with post-mortem or resected brain tissue from a PMS patient with a terminal deletion greater than 5.81 Mb. Perhaps the answer can come from simply comparing cortex samples from patients with terminal deletions smaller than 5.81 Mb and larger. Brain donations to brain banks by patients with rare diseases are a rare and important resource for this type of work.
The third unanswered question is how can loss of a PHF21B gene be rectified? I will not speculate on what approaches might be used to compensate for the loss of a gene so crucial to neurodevelopment. How to fix the loss of neurodevelopment genes is a central problem for all neurodevelopmental disorders. You can try to fix the gene or you can try to compensate for its absence. Perhaps this topic will be the subject of a future blog.
David enjoys walking, but his balance and coordination are not good. He has taken many falls in his lifetime. Sometimes he hits his head. He has had numerous stitches over his eye, on his forehead and has injured his nose more than once. I have always worried that these falls might have a cumulative effect on his brain.
In 2019, just before COVID-19 started to spread, a group of researchers in Shandong, China asked the question: what does the gene CELSR1 do? CELSR1 is on my list of high importance genes of PMS (see Which PMS genes are most important?) One copy of CELSR1 is missing in about half of all people with Phelan-McDermid syndrome who have a terminal deletion of chromosome 22. Only a few months ago I wrote about the relationship between CELSR1 and lymphedema (see Have we found the gene that causes Lymphedema?), based on research done elsewhere in China. As I describe in that blog, research on CELSR1 has become truly international.
It has been known for years that the CELSR1 gene is especially active (producing the celsr1 protein) following a brain injury. The celsr1 protein is associated with the creation of new neurons and enhancing the blood flow in the brain. Until now, it was not clear if the increase in celsr1 protein following injury protects the brain. Now we know a lot more. I will describe the research (see the scientific paper here).
The researchers used a very modern technique. They used a “short hairpin RNA” (shRNA) to reduce protein production by the CELSR1 gene in rodents. This method cut the level of the celsr1 protein production by 50%. Humans missing one copy of the CELSR1 gene should also have about a 50% loss in protein production. In other words, this is an animal model of PMS!
Using a standardized procedure to invoke a brain bleed in rats, the researchers looked to see if rats lacking half of the normal celsr1 sustained more brain damage than expected. Both the amount of brain damage and the ability to walk were significantly affected by the reduced celsr1. The researchers then refined their methods to analyze exactly how celsr1 was protecting the brain. Celsr1 is an amazing protein. It protects injured neurons from dying, it speeds up the production of new neurons and it promotes the formation of new blood vessels to provide oxygen to the injured region. The researchers were also able to pinpoint what part of the brain jumps into action when the celsr1 is needed.
For years, scientists have been looking for natural substances that might protect the brain after a bleed. These “neuroprotective” substances have been studied and they include familiar chemicals, like IGF-1. Thus far, none of the known chemicals or their derivatives have provided useful treatments for brain injury, intellectual disability or autism. The question becomes, is celsr1 nature’s way of protecting the brain? Perhaps we should be focused on restoring the normal level in people missing the CELSR1 gene?
We know that, in general, people with PMS who have larger than average deletions (average size is about 4.5 Mb) have more deficits/problems than people with smaller deletions. This has been shown repeatedly. What we do not know is whether people with deletions greater that 4.5 Mb accumulate brain damage over time. The brain bleeds (strokes) in the rat study are severe enough that a few of the animals die from the bleed. But, are smaller events — perhaps events that David experienced — causing serious long term problems because of the missing CELSR1? If these problems accumulate, perhaps the deletion size will be more important in older people with PMS.
This new information about CELSR1 reminds us that with each important gene comes an opportunity to help people with PMS. Some of the important genes remain poorly characterized. As we are seeing with CELSR1, the more we study these less-well-known genes, the more opportunities we have to develop new treatments.
David loves to walk. He has a strange cycle of activity and there are periods where he just cannot sit still. So, he walks and walks to burn off the need to have activity. Getting him to walk was a major undertaking. It took 6 years of hard work with the physical therapist and his family at home until walking emerged. His gait is clunky. He walks as if he has weights in his shoes. But, he walks and walks when he needs to. Walking is so important for David in so many ways.
David might not be able to walk if he had serious lymphedema. David has peers with 22q13.3 deletion syndrome (Phelan-McDermid syndrome, PMS) who suffer from lymphedema. Lymphedema is seen with PMS Type 1 (and possibly Type 2), but never Type 3 (see The four types of Phelan McDermid syndrome). Undoubtedly, there is a gene somewhere in the list of PMS genes that causes lymphedema (see How do I know which genes are missing?). A group of medical researchers in China who study lymphatic surgery and pediatrics teamed up to treat a 20-year-old woman with lymphedema due to PMS. After careful examination of the woman, her genetics and the pathological mechanisms that can lead to lymphedema, the research group has come up with a very plausible explanation.
The key to this investigation is that the young woman suffers not only from lymphedema, but also from protein-losing enteropathy (PLE). PLE is a condition where the body has an insufficient amount of protein. PLE is related to lymphedema when problems with the lymph vessels supplying the lining of the intestine cause poor absorption of nutrients. When improperly formed intestinal lymph vessels or blockage of lymph flow from the intestines (called lymphangiectasia) occurs, you can have insufficient protein absorption. In 2013 it was shown that loss of the gene celsr1 in mice leads to improperly patterned lymph vessels and lymph valves. In 2016 it was shown that an error in the CELSR1 gene in several generations of a family caused inherited lymphedema in their legs (Gonzalez-Garay et al 2016). Through this understanding, Xia and colleagues have provided a strong case for human CELSR1 as the causal gene for lymphedema in PMS (Xia et al 2021 Lymphedema complicated by protein-losing enteropathy with a 22q13.3 deletion and the potential role of CELSR1).
The researchers were not able to specifically demonstrate GI lymph flow was disrupted in their clinical case, partly because the young woman was not a candidate for lymphography and there was no clinical justification for sampling with endoscopy. Still, the lymphedema was quite clear in this patient and likely the cause of PLE. More importantly, the patterning gene CELSR1 can explain both lymphedema and PLE.
So far as seen the in literature, PLE seems to be very uncommon in PMS. Maybe it showed up in this case because of a diet less protein-rich than in many Western countries. Would we have discovered this sooner if we had more low-income or otherwise disadvantaged people in our community? Diversity and inclusion is not just an ethical concern. It is at the heart of scientific understanding.
Perhaps it is time to look more closely at protein absorption in people with PMS. Are there subclinical cases of malabsorption? If CELSR1 is the smoking gun for lymphedema, people with PMS Type 1 deletions (terminal deletions) greater than 4.3 Mb are at risk for lymphedema and (perhaps) PLE. In both cases, the penetrance is incomplete: the deletion of one copy of CELSR1 makes someone more prone to these problems. We should not dismiss the importance of this work even if the problems are not expressed in every case.
I see an additional take home message from this research. It is clear that helping people with PMS requires understanding and considering all the genes lost when a deletion occurs. This requires input from many different medical and research fields. As parents of children with PMS, it is our job to encourage and reward novel and valuable contributions. In science, one way to acknowledge the contribution of a fellow scientist is to cite his/her work. This blog serves as a thank-you to Song Xia, Wenbin Shen and colleagues. This blog is not the first time I have acknowledged the work of Gonzalez-Garay et al in the Sevick-Muraca laboratory. The mouse work on the Celsr1 protein was done in the Makinen laboratory in the UK and the mouse model was developed by Fadel Tissir in Belgium. Fadel Tissir has studied Celsr1 over many years. All of these people deserve recognition for their contributions. I have personally emailed Dr. Tissir to thank him for his contributions to PMS. Letters of encouragement are a powerful and largely untapped tool we parents have to accelerate the discovery of new treatments for our children. I think we miss an opportunity each time a relevant paper gets published without thank-you notes from our community.
After struggling to learn to read genetic reports with help from generous people like Andrew Mitz, I thought it would be good if other PMS parents could get a little help reading them, too. So I got together with Andy who helped me write this beginner’s guide to reading genetic reports. Click hereto see it. Thanks to Katy Phelan for casting her eyes over an earlier draft of the guide, and improving it. Any mistakes are my own. I will be updating it over time, adding, correcting and responding to suggestions.
Here is a little more of my story.
Our dancing girl didn’t get her PMS diagnosis until she was 16 years old, by which time life had gotten pretty complicated. Even though I’m a pediatrician and child psychiatrist, I didn’t understand the technical language or the real-world meaning of her genetic report, and our meeting with the geneticist wasn’t all that much help.
PMS Foundation staff helped, and so did Alex Kolevzon, Andrew Mitz, and Catalina Betancur, each of whom reviewed our girl’s testing with me at different points in the following two years. I went on to talk with many parents, and to do research on psychiatric illness in PMS, which required learning more about variants, small deletions and big deletions.
Four years later I am still learning, and the field continues to grow. Families now turn to me for help understanding the meaning of their child’s report. And when I am not sure, I still turn to Andy, who’s been at this a lot longer than I. Andy and I know how overwhelming it can be to look at these reports that frame our children’s lives. I hope this will give parents a head start for their conversations with genetic counselors and geneticists.
Originally created 12 September 2020
Updated 31 January 2022
I am going to describe four types of Phelan-McDermid syndrome (PMS). By type, I mean four different genetic arrangements that can result in PMS. These are technically called “genotypes”. The four genotypes do not address the mechanisms that create each genotype. For example, my son, David, has Type 1 (Genotype 1) from an unbalanced translocation, but others have PMS Type 1 from de novo deletions or ring chromosomes. The types can be used by parents as a very simple short-hand to share information about their child.
Phelan McDermid syndrome Type 1, terminal microdeletion
This is the original 22q13.3 microdeletion syndrome described by Katy Phelan and Heather McDermid. It is also the most common type among people identified with PMS.
A microdeletion occurs when multiple genes on a chromosome are removed. Most microdeletions are too small to see under a microscope, but Katy Phelan has described some microdeletions that are large enough to be visible. Deletion sizes range from about 0.2 Mb to 9.3 Mb, with an average of 4.5 Mb. Nearly all of the microdeletions observed are terminal deletions (continue to the end of the chromosome). Terminal deletions much smaller than 0.2 Mb do not include SHANK3 and do not produce a syndrome.
Phelan McDermid syndrome Type 2, interstitial microdeletion
An interstitial microdeletion is a deletion that removes multiple genes, but does not extend to the end of the chromosome. For PMS Type 2, the deletion does not reach SHANK3, which is near the end of the chromosome.
Interstitial deletions that produce PMS, but do not disrupt SHANK3 are relatively rare. Some scientist have argued against including them in the definition of PMS. However, a recent scientific consensus paper (https://ojrd.biomedcentral.com/articles/10.1186/s13023-022-02180-5) justifies the strong reasoning for including Type 2 in the definition of PMS.
Phelan McDermid syndrome Type 3, single SHANK3 rare variant
A rare variant is an atypical version of a single gene. PMS caused by a rare variant of SHANK3 is called Type 3. Sometimes this has been called a “mutation” of SHANK3. That terminology is not always technically correct, so “variant” is preferred.
Type 3 is currently the second-most common example of PMS. However, rare variants of SHANK3 have been found in large collections of DNA from people with autism spectrum disorder (ASD). Although only 1 to 2% of people with ASD might have a rare variant of SHANK3, this still a potentially large population of people who might have PMS Type 3. More research is needed.
Phelan McDermid syndrome Type 4, heterozygous deleterious variants
You child does not have PMS Type 4. PMS Type 4 is included here for completeness. Individuals can inherit two copies of unusual variants of SHANK3, one from each parent. This can produce PMS as a recessive disorder. There may be cases of Type 4, but they would be very rare, indeed.
The consensus paper (see the link, above) has provided a clear statement about how the PMS diagnosis should be applied. This is the classification that geneticists and scientist should use.
PMS-SHANK3 related (Types 1, 3, 4)
PMS-SHANK3 unrelated (Type 2)
Note that most cases of PMS fit one of the four PMS types. However, the reality of genetics is that some cases can be very messy. The four genotypes of PMS are not always clearly segregated in any given individual. An individual can have an interstitial deletion, yet also have a pathological variant of SHANK3. This could be described as both PMS Type 2 and PMS Type 3. The classification system from the consensus group, PMS-SHANK3 related versus PMS-SHANK3 unrelated,covers all possible cases without any overlap.
Sometimes two different genotypes are actually very similar
Most PMS Type 1 and PMS Type 3 cases are very different from each other. An individual with a large terminal deletion is likely to have problems not shared by an individual with a SHANK3 variant. With Type 1 there are many important genes involved, some known to produce profound effects. In Type 2, any effect would only result from disturbing SHANK3. Yet, someone with a small terminal deletion (PMS Type 1) might be very similar to someone else with PMS Type 3. Although in different categories, they could end up being very similar.
PMS is a syndrome
PMS is defined by its genetics. However, central to all syndromes is that the individual must have a phenotype reflecting the syndrome. There are some individuals with interstitial deletions and other individuals with with SHANK3 variants who simply do not have significant features of Phelan-McDermid syndrome. These are very interesting cases, but they cannot be called PMS.
This simple system of PMS Types 1 to 4 can help parents quickly share the basic genetics of their children. PMS Types 1 and 2 have a deletion size associated with them. PMS Type 3 does not. Some manifestations of PMS, like lymphedema are not seen with Type 3. Sharing your child’s PMS Type can be an ice breaker during introductions and help a parent share key information without having to be a genetics expert.
It has been known for some time that many people with Phelan McDermid syndrome (PMS) have mitochondrial issues. I discussed this in an earlier post (see Is 22q13 deletion syndrome a mitochondrial disorder?). At the time of that posting there were 17 PMS genes known to impact mitochondria. There is evidence that the PMS gene RABL2B generates a protein to transport mitochondria into synapses. That would be 18 genes. Now, new evidence has emerged that the SULT4A1 gene, a highly important PMS gene (see Which PMS genes are most important?) is critical for protecting the brain from oxidative stress by regulating mitochondria function.
After 20 years the role of SULT4A1 is finally coming to light. SULT enzymes have been known as important enzymes for a while, but SULT4A1 has always been a mystery. The other SULT enzymes have an active region used to regulate critical proteins in the cell, some involved with mitochondria function and the key neurotransmitter, dopamine. But, the SULT4A1 protein lacks the same active enzyme site. The other mystery has been that the SULT4A1 gene is highly specific for brain and brain development. Cathrine Ziats’ scientific paper last year found SULT4A1 to be one of the top 4 PMS genes expressed in the human brain during development (see her paper: Functional genomics analysis of Phelan-McDermid syndrome).
The new evidence paints a picture placing SULT4A1 as a critical regulator of brain metabolism. The thing to understand about the brain is that it sucks up 30% of the total body’s energy supply! This puts a huge load on the mitochondria of the brain. SULT4A1 regulates two other SULT proteins, SULT1A1 and SULT1A3. These enzymes are found on the outside membrane of mitochondria, connected together in pairs. By regulating these enzymes, SULT4A1 is able to crank up the output of the brain’s mitochondria. This reduces the reactive oxygen species, reduces oxidative stress and prevents neuronal damage. (See the two articles on PubMed: Hossain et al 2019 and Idris et al 2020). As SULT4A1 regulates these two other enzymes it can also regulate the effects of dopamine. Dopamine is a key neurotransmitter involved in learning and decision making. Too much dopamine, especially during development, can damage a cell. Too little dopamine is associated with motor and psychiatric disorders, like Parkinson’s disease and major depressive disorder.
Nearly a third of our PMS kids are missing the SULT4A1 gene (deletions larger than 7 Mb). Finding a way to fix the SULT4A1 gene would be a game-changer for these children. Like the other essential brain genes of PMS (e.g., SHANK3), the precise regulation of SULT4A1 is critical to normal development and healthy brain function. There are people with interstitial deletions of 22q13 that impact SULT4A1 without affecting SHANK3, and these people have severe developmental problems indistinguishable from others with PMS. (That some scientists are still arguing over whether or not to keep them in the family of PMS is a travesty, in my opinion as a father. See PMS, IQ and why interstitial deletions matter.)
We need more research on SULT4A1. We need treatments sooner rather than later.
Phelan McDermid syndrome (22q13.3 deletion syndrome, PMS) comes in three flavors. By far, the most common variety of PMS that has been observed is a terminal microdeletion. A terminal deletion occurs when from three to 108 genes are chopped off the end of chromosome 22. There is a growing group of identified patients who have structural changes restricted to the SHANK3 gene, a gene very near the end of the chromosome. These changes can be minimal (e.g. single nucleotide polymorphism (SNP)) or substantial, with large parts of the gene duplicated or missing. The least studied form of PMS is found among those deletions that damage the same general region of chromosome 22 without affecting the very end of the chromosome where SHANK3 resides. These are often called interstitial deletions.
Genes of PMS
The SHANK3 gene is an elephant. It is a large, complicated gene and its disruption often (but not always) has severe consequences for its owner (the person harboring the mutation). Numerous studies show that the many genes of 22q13.3 contribute to intellectual disability, muscle tone and movement disorders, hearing problems, lymphedema, autism, schizophrenia and other problems. But, the penetrance of SHANK3 mutations, that is the likelihood that a pathological variant of the gene affects the owner, is high. So, it garners a lot of attention. Especially for people with the second flavor of PMS, fixing SHANK3 could ameliorate many of the symptoms associated with its disruption. For terminal deletions, the most common form of PMS, fixing SHANK3 should have at least a very helpful impact.
There are at least 18 genes that may need repair to fully restore normal function in a child with a large terminal deletion. Average size deletions (about 4.5 Mbase) would benefit from the repair of 10 genes. This blog discusses what we hope will be the future of PMS treatment: genetic repair. While regular medical treatments have been helpful for some problems, no experimental drugs have had an impact any better than physical therapy, speech therapy, ABA or other standard treatments for children with developmental disabilities. Although drug companies are sponsoring testing on children with PMS, these have traditionally been drugs looking for an application rather than targeted treatment for PMS.
We are in the early days
Excitement over gene editing, especially in stem cells (cells typical of a very early embryo) has led to giant research efforts and early attempts to fix diseases by editing. The same excitement has led to the promotion of unproven and occasionally dangerous therapies by entrepreneurs who provide services like stem cell infusion. So, let’s be clear. Gene therapy for PMS is well off into the future. There are many reasons for the delay. Some will be discussed below.
Obviously, we are at the earliest stages of gene therapy. Research and initial attempts at gene therapy target diseases that are the safest and most likely to work. This means that the disease most likely to be cured first may be a rather obscure one. It may also be a lethal disease, since the risks of side effects are no worse than letting the disease take its course.
One relatively safe way to test gene therapy is to modify blood products and then infuse them back into the body. This has safety advantages in a few different ways. First, the genetic modification occurs outside the body. The target cells can be modified without risking other cells of the body. Second, the target cells can be tested and evaluated for successful gene targeting (editing the right spot and not accidentally damaging another gene) outside the body. Third, the product can be infused a little at a time to test the benefits/impact gradually. Fourth, blood products usually have a limited lifetime in the body, so if the test begins having problems, the problems will likely go away with time. Thus, gene editing for diseases of the blood are good early candidates.
Another target for early genetics testing is the eye. It is a squeamish thought, but making injections into the eye are relatively easy. (I have had eye injections, so no need to educate me on the downsides.) The inner parts of the eye are well isolated from the rest of the body, which affords safety. Also, only very small injections are needed to bathe the retina. There is little likelihood of impacting the rest of the body. The amazing thing about eye therapy is that modern cameras and computers can take images of the eye with spectacular resolution. These images can track changes. Combined with vision testing, any progress associated with the therapy is easy to assess. As gross as it sounds, if something goes wrong with gene editing of the eye, the eye can be removed. Gene therapy for the eye would be to save vision. If vision will be lost with no intervention, the risks of trying a new therapy may be acceptable. Thus, genetic defects of the retina are high on the list of early therapeutic trials.
The human brain is not well isolated from the rest of the body and is not easily observed from the outside. There is a barrier between the bloodstream and the brain. The so called blood-brain-barrier can make treatment more difficult. Drugs injected into the bloodstream (systemic injections) may or may not reach the brain. Direct brain injection of genetic altering “vectors” may be an approach used in the future, but it is not ideal. Systemic injections would be a simpler way to reach all parts of the brain and to allow repeat injections to gradually reach the desired effect while watching for side effects. So, genetic editing in the brain is not going to be as simple as other targets.
Once we start thinking about genetic manipulations in the body, we need to remember that nearly every cell in the body has the same 20,000 genes. Given that all cells in the body could be the same, what makes heart and lungs different from kidney and brain? The answer is gene expression. Heart cells know they are heart cells because only heart cell genes are turned on, making the proper heart cell proteins in the proper proportions. We hope that injecting a vector into the body will improve brain function, but we must worry about what else may change. We worry in two ways. First, we could modify our target gene in such a way that the brain improves, but the heart (or some other organ) suffers. Second, we could accidentally modify other genes (so called, off target effects) that have no adverse impact on brain cells, but might be bad for the heart. I used the heart as an example, but there are so many different tissues in the body we cannot know which might be adversely affected.
One more consideration is important in choosing a target for the early tests of genetic therapy. The complexity of the organ and the importance of early development in creating its detailed structure influence the age at which therapy may be effective. The brain is complex almost beyond comprehension and its early patterning during development is critical to adult function. Repair of a gene after early development may have limited benefit, and except for the most deadly of disorders, we dare not test interventions on fetuses or babies until we are confident of our methodology. Additionally, the complexity of the brain poses another risk. Increasing the production of a gene may be beneficial for one part of the brain, but could be detrimental elsewhere in the brain.
The challenges of PMS
The opening of this blog hinted at some of the challenges of PMS. Together with the subsequent discussion, we can now list the major challenges of applying early genetic methods to PMS: 1) the overwhelming majority of people diagnosed with PMS are missing many genes and modern methods are still struggling for success with single genes, 2) the gene of greatest interest is large and complex, neither of which is good for gene editing, 3) the brain is a challenging target for genetic manipulation, and 4) PMS is a developmental disorder and we have insufficient information on whether treatment in older children or adults will be beneficial.
Given the many hurdles, it may be a very long time before a comprehensive treatment for PMS emerges. To be sure, there are people working on strategies to manage the disadvantages. One approach is to target only one gene in the hopes that at least some subset of patients might benefit. Likewise, there are model (rodent) experiments that suggest some of the deficits of PMS can be mitigated by treating adults. Genetic treatments for other diseases are addressing the problem of reaching the brain without adversely impacting other organs. Both animal and early clinical trials are creating improved toolboxes for genetic therapies. We hope these technologies converge on better opportunities for PMS.
Progress in the field
Techniques like CRISPR/Cas9 grew out of studies in bacteria and other science far afield from human clinical work. Recent work in E. Coli and other bacteria, as well as work on yeasts and viruses have moved genetic editing towards more precise targeting, the possibility of replacing whole deleted sections of chromosome, and the ability to enhance the activity of the remaining gene after one has been deleted. There are new methods of turning on and off a newly inserted gene, so the proper dose of a gene can be safely titrated to avoid overdose. This blog has not addressed the different methods of gene editing and which ones might apply to PMS. Rather, it provides a framework to understand why one approach or another might be more suitable. Perhaps a future blog will cover the different tools and which ones are likely to provide the first clinical tests.
A strategy for PMS
After reading the scientific literature and talking with scientists, I have a policy suggestion for how we should approach PMS. Currently, the only genetic intervention being pursued for PMS is targeted at the SHANK3 gene. There are historical and logical reasons for targeting this gene. It is highly associated with several developmental disorders. That work should continue, but from a technical perspective, SHANK3 is not an easy gene to treat. The SHANK3 gene can produce from 20 to 100 isoforms (versions) of the shank3 protein, up to 1731 amino acids in size. (By comparison, the UBE3A gene associated with Angelman syndrome produces a maximum of 12 ube3a isoforms, with 875 amino acids being the largest. The MECP2 gene associated with Rett Syndrome, has two isoforms, with 486 amino acids being the largest.) Manipulating a large, complex gene like SHANK3 has consequences, at least some of which will likely be negative consequences. Like all early genetic clinical studies, initial trials will be on a few hand-picked cases. Unfortunately, even among people with SHANK3 mutations, there are many flavors and early treatment methods might not generalize to many patients.
If we are serious about a long-term solution for PMS we need to work on a smaller and more simple gene. Solving problems caused by SHANK3 is going to be a very slow process. We should choose a gene much easier to fix, and yet still has a serious impact in our children with PMS. The idea is to bring relief sooner, and to begin the important task of treating the full spectrum of PMS. There are 17 possible genes, three or four of which are among the best candidates. One possible target is BRD1. This is a regulatory gene that has been very well characterized. It is smaller than SHANK3 and one copy is missing in about 90% of the PMS population. Evidence so far supports the importance of missing only one copy of BRD1.
Titrating expectations, but not hope
There is always a balance between the enthusiasm for research into effective treatments and maintaining a realistic view of scientific progress. Young parents have so many compromises to make in their hope for the future when they receive a diagnosis of PMS. Placing the disorder into perspective takes years. We grow into a life consumed by a child with a major developmental disability. Hope requires embracing the possibilities, maintaining enthusiasm about the future, yet titrating our expectations to match today’s realities. We are a long way off. Our own children will likely be adults, perhaps old ones, before effective genetic treatments are ready for widespread application. We need to be realistic about our expectations without diminishing our hopes. Indeed, this is what raising a child with PMS is all about.
Originally posted 30 December 2019
Updated 4 March 2022
Regression is one of the scariest words among the Phelan McDermid syndrome (PMS) community. It is not clear how common regression is in PMS. Recent and ongoing studies are addressing the problem. Regardless, regression does occur, can be unpredictable, and it can sometimes be devastating. After years of hard work helping your child navigate a world that is well beyond his or her comprehension, you then lose some or all of the gains to an invisible and unforgiving demon. In the PMS world, you either loath regression or fear its occurrence.
Three papers recently looked at regression in PMS, each taking a somewhat different approach. Reierson et al 2017 used scores from the Autism Diagnostic Interview-Revised (ADI-R) based on 50 patients seen in their clinical study. Verhoeven et al 2019 studied 24 adult PMS patients with psychiatric presentations and followed them over years. Kolevzon et al 2019 did a detailed literature review of PMS and SHANK3 mutations (denovo deleterious variants). This blog will be mostly a technical overview of the Kolevzon paper, with some additional notes from the Verhoeven study, both aimed at patients over 12 years of age and who present with psychiatric issues or sudden loss of function.
All of the authors have struggled to define “regression” and identify trends in occurrence and treatment methods. I am told there are important differences between “regression” and psychiatric decompensation. I’m not a psychiatrist so I won’t untangle the technical differences. But from what I read, either type of event can be a setback. Psychiatric decompensation seems to bring with it the likelihood of recovery and can repeat (episodes).
If you don’t want to read my full post, here some of the take-home messages.
1) There is yet no clear measure of how often regression or psychological decompensation occurs with PMS.
2) Relying on previous papers is difficult because not all descriptions in the literature provide sufficient detail about each patient.
3) Diagnosis of psychiatric illness is more difficult in people with low intellectual function and absent of functional language.
4) People with SHANK3 variants (“mutations”) and tiny chromosome deletions are a small minority of identified PMS cases. In general, they are more functional than others with PMS (higher intellectual function, better language and better motor skills). This minority are the patients who most often present with psychiatric problems. The great majority of patients with deletions greater than one or two Mb (more common PMS patients), show psychiatric problems far less often, at least given the limited research done so far.
5) When looking at the psychopathology overlap between SHANK3 variants and typical PMS patients, the common feature seems to be bipolar disorder.
6) In spite of the difficulties studying psychiatric problems in PMS, following best practices of psychiatric care used in the general population can be helpful for people with PMS.
7) Over-medication and certain medication choices can harm, rather than help.
8) Proper personal and social care of the adult with PMS is important for the successful management of regression and psychiatric problems.
9) These early studies provide a first, and perhaps hopeful picture, but much more work needs to be done to understand the natural history of PMS and the risks we face as parents.
Most of the important information from the Kolevzon paper is in their Table 1, which is the result of an in-depth search for papers on PMS patients. Table 1 provides an excellent resource to the PMS research community despite ignoring interstitial deletions. I spent a few hours hunting down the original articles listed on the table and studying them. PMS science benefits greatly from this work. The table itself has a flaw that readers should be aware of. The reference numbers of the table are messed up. The following reference numbers are wrong (4-6, 8-12, 15-19, 22-25, 30, 32, 35-42). The author names and dates are correct, so there is no problem if you simply ignore the reference number and do a text search for the author name.
As noted above, it is difficult to define regression and the authors did not clearly describe the difference between regression and psychological decompensation. Presumably, experts in the field recognize the distinction. Regardless, the Kolevzon study required some rule set to execute a literature review. These authors chose: “Sudden change in the psychopathological presentation”. They focused on patients older than 12 years. The literature search looked for: 1) psychiatric decompensation, 2) loss of skill, 3) sudden behavioral change.
They found 56 cases, including 15 cases of ring chromosome, referred to as r(22). While it is laudable to dig deep into older medical literature and pull out r(22) cases, most of these predate the time when deletion size was easy to measure. There is another problem with evaluating r(22). Just as interstitial deletions can cause PMS without disrupting SHANK3, r(22) can cause developmental delays without disrupting SHANK3. In fact r(22) can cause developmental delays without disrupting any gene (see: Guilherme et al 2014). Also, as the Kolevzon paper points out, r(22) can lead to Neurofibromatosis 2 (NF2) disorder, which can be degenerative. The authors are careful to identify which results are dominated by r(22) cases. I think only the most recent cases (cases 19, 35 and 51) are helpful to the study. The earlier cases date from 1985 to 2007. One other case, 32, does not seem to provide much information.
By removing cases of PMS deletions that lack deletion size information, we can explore the impact of deletion size. What we discover is that psychopathology is almost exclusively diagnosed in patients with small deletions. The plot below uses the Kolevzon data from Table 1. It shows the frequency of diagnosis (number of cases) as a function of deletion size. SHANK3 variants are not included.
To understand these results, remember that 95% of people identified with PMS have deletions greater than 1 Mb (see: Understanding deletion size). The cases found by Kolevzon are heavily weighted towards small and very small deletion. That is, most cases of psychopathology in the literature come from deletions that represent about only 5% of the PMS population.
If we merge the data from SHANK3 variants with the smallest deletions (size 0 to .9 Mb), the first bar more than doubles in size. We can conclude that psychopathology shows up almost exclusively when chromosome 22 is disrupted near the terminal end. That is, it shows up in the atypical cases where damage is limited to primarily SHANK3 disruption. Is this generally true for PMS or just for the sample of papers found during the literature search? We don’t know. For the moment, it is the best data available.
The authors suggest that because people with SHANK3 variants and tiny deletions are higher functioning, psychopathological changes might be easier to observe. Indeed, in their sample the cases of SHANK3 mutation and small deletions had both significantly higher intellectual function and motor skills. I suggest another possibility. It may be that loss of genes in the 2 – 4 Mb region of the chromosome help stabilize brain function. The most likely candidates in this region of the chromosome are BRD1, TBC1D22A, GRAMD4, CELSR1. I have discussed some of these genes in detail, in my other blogs (see 22q13: a hotbed for autism and intellectual disability genes?, and CELSR1: Do some people with PMS have more fragile brains?) Whether these genes seriously impact brain function, stabilize the brain, or both, there is no doubt that these genes greatly influence the outcomes of patients with PMS. The importance of these genes have come up in other studies, as well. These genes likely also contribute to the impact of PMS from interstitial deletions (see: Which PMS genes are most important?).
These initial results are concerning to families dealing with very small deletions or SHANK3 variants (limited primarily to SHANK3 disruptions). On the other hand, most of the Kolevzon study has less relevance to 95% of families. Still, there are 8 of the 56 patients that represent typical size deletions: P20, P21, P23, P24, P28, P33, P47 and P50. It is valuable to look specifically at these typical cases of PMS to see what might be more generally relevant to PMS families. For example, none of these 8 patients were diagnosed with psychosis and only one had tremor. Thus, psychosis and tremor might not be very relevant to most families. On the other hand, only typical PMS patients lost the ability to walk. Catatonia was seen in only one typical PMS patient (P50).
One feature in common between typical PMS patients and the uncommon cases of SHANK3 disruption is the incidence of bipolar disorder. Half of the typical PMS patients were diagnosed with bipolar disorder, as were many of the patients with SHANK3 disruption. This observation suggests a common mechanism, likely the SHANK3 gene. Do PMS patients with interstitial deletions have bipolar disorder? That would be an extremely valuable question to explore. Reviewing cases of interstitial deletions may support or refute the specific role of SHANK3 in bipolar disorder.
Verhoeven et al observed a similar overlap between typical PMS and cases of SHANK3 disruption. They state: “Based on actual psychiatric classification, in 18 patients, a diagnosis of atypical bipolar disorder was established of which symptoms typically started from late adolescence onward. In most patients, treatment with mood stabilizing agents in combination with individually designed contextual measures, and if indicated with the addition of an atypical antipsychotic, resulted in gradual stabilization of mood and behaviour.” Note the phrase “with individually designed contextual measures”. Proper social treatment of the person with PMS appears to be important for “recovery” (not a technical term).
Kolevzon et al discuss which treatments were more or less effective for both the SHANK3 disruptions and typical PMS patients. The overlap with Verhoeven is very encouraging. Together, the agreement between recent studies on regression and psychopathology provide improved guidance for the medical caregivers who may encounter bipolar disorder in PMS patients. The work of Kolevzon is especially valuable for cases of SHANK3 disruption (variants and very small chromosomal deletions). The number of cases where the PMS adult was stabilized was encouraging in both studies, which provides hope for families facing adult psychopathology.
It will take more studies to make strong statements about regression and psychological decompensation in Phelan McDermid syndrome. These early studies hint at the type of problems that do occur in PMS, the types of remediation that have been beneficial and the differences that appear to be associated with deletion size.
My son, David started life with some rather major problems. He was 6 years-old before he could walk and 9 years-old before he could eat by mouth. He never developed speech. His developmental trajectory has been slowly upward. At age 34, he has not had a regression or anything that could be called psychological decompensation. (His mom is qualified to recognize decompensation.) His deletion size is unknown, but I would wager it is not a small deletion. Does his stability come from missing certain genes?
When we moved David to a new group home two years ago, his mom and I were vigilant about managing the changes and constantly gauging his comfort. We were, frankly, scared that he would decompensate or regress. The transition was successful and David feels at home. But, what will be the next challenge and how will David respond? We don’t know.
While SHANK3 has been identified as a major contributor to intellectual disability and autism spectrum disorder, there has always been a lingering question of why people with Phelan McDermid syndrome (PMS) have very similar symptoms (phenotype) whether or not SHANK3 is impacted. That is, people with terminal deletions that remove SHANK3 are not very different from people with so-called “interstitial deletions”. This observation is the main reason why the current definition of PMS says that SHANK3 is usually, but not always, impacted (see The 22q13.3 Deletion Syndrome).
Genes with a high “pLI” (genes that are very rarely mutated in the general population) are associated with intellectual disability. For 22q13 they are: SHANK3, MAPK8IP2, PLXNB2, TRABD, PIM3, ZBED4, BRD1, TBC1D22A, GRAMD4, CELSR1, SMC1B, PHF21B, PRR5, SULT4A1, SCUBE1, TCF20, SREBF2, and XRCC6. Most, if not all of these genes contribute to intellectual disability, although some are more certain. While 18 genes may seem like a lot of genes, remember that PMS can delete up to 108 genes. Still, it is impressive that nearly 17% of the genes of 22q13 potentially contribute to intellectual disability. The high number of these high pLI genes explains why intellectual disability is generally more severe with larger deletions, and it explains why interstitial deletions can also produce moderate to severe intellectual disability. But, what about autism spectrum disorder (ASD)? The frequency of ASD in PMS is an area of debate, various studies report anywhere from under 30% to over 70% incidence of ASD. The natural history study of PMS should narrow this range. Regardless, ASD is a component of the PMS population.
So far, three genes of 22q13 have been clearly associated with ASD: SHANK3, MAPK8IP2 and SULT4A1. It is probably no accident that these three genes also show up as high pLI genes.
What is new
Two new lines of research have identified new candidate ASD genes on 22q13. One candidate gene has arisen from studies of epigenetics, the regulation of genes. Genes occupy only about 1% of the DNA. Much of the remaining DNA is dedicated to controlling when and how often a specific gene is put to work. Gene regulation is essential for successful development of the brain (and all other body parts) and for responding to changes in the environment. By environment we can refer to the external environment (e.g., adapting to colder weather) or we can be concerned with the internal environment of the body (e.g., building muscle with increased exercise). There is earlier data suggesting a weak contribution of the gene BRD1 to ASD (see Which genes cause brain abnormalities in Phelan McDermid syndrome?). However, epigenetic studies have reinforced the connection between BRD1 and ASD (see Next-gen sequencing identifies non-coding variation disrupting miRNA-binding sites in neurological disorders). So, BRD1 can be considered an autism-associated gene.
Perhaps more exciting is the latest work on “second hits”. Second hits are when one gene is lost or damaged, and then the remaining copy of the gene is also affected. Second hits matter because some genes can operate just fine with only one copy. We have two copies of most genes in our DNA, one from each parent. When a person has a chromosomal deletion, which is what happens in over 95% of the cases of PMS, that leaves the person with only one copy of those genes. A second hit is when the remaining copy has a mutation, or even just an dysfunctional variant inherited from one parent. What do we know about second hits? A new paper has identified up to 4 genes of 22q13 that may contribute to ASD, given a second hit (see Recessive gene disruptions in autism spectrum disorder). One PMS gene stands out, KIAA0930 and three others are additional candidates, CHKB, ARFGAP3, and ZBED4.
This rounds-out the current full list of 8 ASD-associated genes: SHANK3, MAPK8IP2, CHKB, ZBED4, BRD1, KIAA0930, SULT4A1, and ARFGAP3.
PMS is most often a terminal deletion of 22q13. A deletion of the chromosome that includes SHANK3 is the most common deletion, but sometimes a deletion does not disrupt that gene. Interstitial deletions can disrupt up to 17 other genes likely to contribute to the moderate to severe intellectual disability that characterizes PMS. Infrequently, a second hit on one or more of 7 genes in the interstitial region of 22q13 may also contribute to autism spectrum disorder. In the overwhelming cases of PMS (called terminal deletions), many genes contribute to intellectual disability and, if autism spectrum disorder is part of the clinical picture, SHANK3 and one or two other genes may also be contributing to that aspect of the disorder.
For years I have looked for a simple way to explain Phelan McDermid syndrome (PMS) to David’s caregivers. What is it and how does it happen? Here is my latest version.
What is PMS?
Since the overwhelming majority of PMS cases include intellectual disability, we can use the following definition to help explain PMS (see additional note #1). PMS requires:
A chromosome deletion in the region called 22q13, and/or disruption of the SHANK3 gene also in that region.
Intellectual disability (IQ less than about 75 (see additional note #2))
How does the deletion in 22q13 cause intellectual disability? Take the IQ that came from the parents, subtract the IQ loss from the SHANK3 gene disruption and the loss from the other genes that are deleted (see this blog for the science). If the resultant IQ is below 75 or so, the person has PMS.
The main problem with this formula is that we don’t know what IQ a child would have without PMS. Future research might make it possible to know, but we don’t know now. We also don’t know exactly how many IQ points are lost with SHANK3, but we might have that number soon. Even with these problems, we can use the formula to get a better understanding of what causes PMS. Here are some very instructive (made-up) examples.
Example 1: interstitial deletion near SHANK3, but SHANK3 unaffected
Above average IQ from the parents = 116
Loss from SHANK3 = 0 (SHANK3 is not affected)
Loss from 1 Mb interstitial deletion = 25
116 – 0 – 25 = 91
ANSWER: Not PMS (an IQ of 91 is within the normal range)
Example 2: 6 Mb interstitial deletion, SHANK3 unaffected
Average IQ from the parents = 100
Loss from SHANK3 = 0 (SHANK3 is not affected)
Loss from 6 Mb interstitial deletion = 50
100 – 0 – 50 = 50
ANSWER: PMS (an IQ of 50 is mild to moderate intellectual disability)
Example 3: Terminal 1 Mb deletion (SHANK3 is completely removed by a terminal deletion)
Average IQ from the parents = 100
Loss from SHANK3 deletion = 40
Loss from 1 Mb deletion = 25
100 – 40 – 25 = 35
ANSWER: PMS (an IQ of 35 is moderate to severe intellectual disability)
Example 4: 6 Mb terminal deletion (9 Mb is the largest, 4.5 Mb is the average size deletion)
Average IQ from the parents = 100
Loss from SHANK3 deletion = 40
Loss from 6 Mb deletion = 50
100 – 40 – 25 = 10
ANSWER: PMS (an IQ of 10 is profound intellectual disability)
These are not intended to be “real” cases but they could happen and do illustrate the cumulative loss of several or many important genes.
In most cases, PMS is not complicated to understand. From these examples, we get an idea of what must be done to overcome the intellectual impact of the syndrome.
There is no universally-agreed upon definition of PMS, but most PMS researchers readily agree in most cases. It seems possible that someone can have IQ in a normal range, but still have PMS. That might occur if someone has another major feature of PMS, like severe hypotonia or significant expressive language problems.
IQ has different ranges. The threshold for intellectual disability (ID) can be different from the threshold for normal (average IQ). Normal range is above 84. “Borderline IQ is 70-85”. ID is sometimes considered anything below 75, but below normal is anything below 85. This blog is not a diagnostic manual, so no one should use the numbers as a substitute for professional testing, diagnosis or treatment.
No one yet knows the exact loss of IQ associated with SHANK3 deletions The value assumed here is 40, which is not unusual for high penetrance genes. Data from PMS patients, especially data from interstitial deletions, will help determine the exact value.
Note that interstitial deletions can cause PMS, or not, depending on the severity of the deletion. There are a few people who disagree with this part of the definition.
SHANK3 de novo mutations (also called variants) can cause PMS, but there are clear examples where the exact same variant causes PMS in some cases, but not others. This may be the result of the initial IQ of the individual. We assume average IQ of 100 for some of the examples, but actual IQ in the general population can commonly be from 85 to 115. Some SHANK3 variants may have little or no impact on IQ. Other variants may have additional serious consequences (called gain-of-function variants).
Chromosome 22 terminal deletions are obviously quite impactful. They remove all of SHANK3 and lead to major IQ losses from the rest of the deletion. This is how PMS was discovered in the first place.
Brain structure and functional abnormalities have been reported in Phelan McDermid syndrome (PMS) by a number of different investigators, including a a recent study associated with the Developmental Synaptopathies Consortium natural history study of PMS (Srivastava et al 2019). Prior studies have found abnormal formation of the cerebellum (Aldinger et al 2013), abnormal function of the cortex and amygdala (Philippe et al 2008), micropolygyria (Kurtas et al 2018), as well as commonly observed thinning of the corpus callosum and the presence of arachnoid cysts.
Brain abnormalities can provide important clues to understanding what goes wrong in PMS. They also could serve as “biomarkers”, biological measurements for early indicators of severity or evidence for treatment effectiveness. Readers of my blog will recognize that I spend a lot of time identifying which genes are most important in PMS. You need to know which genes are causing what problems to have any hope of finding effective treatments. My son, David (see picture), is waiting for new treatments.
So, what do the brain structural studies tell us?
SHANK3 variants can impact the proper development of white matter, but have minimal impact on the gray matter of the brain (Jesse et al 2020). Gray matter is where the neurons and their synapses reside. White matter is made up of the long, thin “axons” that travel together connecting one region of the brain with another region.
Aldinger and colleagues (Aldinger et al 2013) studied 10 subjects with PMS using x-ray images. Eight of the 10 subjects showed abnormality of the cerebellum (mostly gray matter) in addition to thinning of the corpus callosum (white matter) and enlargement of the cerebral ventricles (fluid space of the brain). Although there was no clear effect of deletion size, mutation of SHANK3 was not sufficient to cause cerebellar problems. They identified MAPK8IP2 and PLXNB2 as the more likely candidates for cerebellar malformation based on preclinical mouse studies.
The study by Philippe (Philippe et al 2008) had similar results from 8 PMS subjects. Three of the 4 subjects with small deletions (150 Kb or less) had no cerebellar or other major magnetic resonance imaging (MRI) results. The 4th subject with a small deletion had the least impressive positive finding. Thus, using MRI, there was a clear effect of deletion size, with small deletions have little or no effect. Four deletions of 1 to 9.3 Mb in size had stronger effects. Like the Aldinger study, SHANK3 did not seem to be a good candidate for most of their findings. In addition to cerebellar malformation, the group studied brain function using positron emission tomography (PET). They showed a group effect of amygdala dysfunction. Importantly, they used children with intellectual disability as their control group, a much stricter standard than other PMS studies.
The Srivastava study (Srivastava et al 2019) showed reduced size of the dorsal striatum, which is the opposite effect that loss of SHANK3 has in mouse models of PMS. Together, the results suggest that genes other than SHANK3 are driving brain malformation. SHANK3 can contribute to thinning of white matter, but is not the cause of other brain malformations.
Which genes might be driving the observed effects? Is there a smoking gun? To be a smoking gun, a gene that drives malformation should meet most, or all, of these criteria:
is strongly associated with a human neuropsychiatric or neurodevelopmental condition
causes reduced brain size in the striatum
impacts the amygdala
There are 6 genes on chromosome 22 that meet criteria 1 and 2. They have a high pLI score and are very frequently lost in PMS. They are located within 1 Mb of the chromosome terminus, which accounts for 95% of patients with a 22q13.3 deletion (see my blog Understanding deletion size). Those genes are: MAPK8IP2, PLXNB2, TRABD, PIM3, ZBED4 and BRD1. Of these, 4 genes meet criterion 3, being highly expressed in the cerebellum: MAPK8IP2, PLXNB2, ZBED4 and BRD1. Of these, 3 genes are associated with neuropsychiatric disorders. BRD1 is strongly associated with and schizophrenia. MAPK8IP2 is weakly associated with ASD (see my blog Which PMS genes are most associated with Autism?), and ZBED4 is weakly associated with schizophrenia. This leaves BRD1 as the strongest candidate gene for brain abnormalities/malformation in PMS.
Animal studies of BRD1 agree with the results from PMS imaging studies. Per Qvist and his colleagues in Denmark have been studying the BRD1 gene for some time. They have shown that loss of one copy of Brd1 in mice is sufficient to reduce cerebellum size, reduce striatum size and reduce the size of the amygdala (Qvist et al 2018). BRD1 is by far the strongest candidate gene for causing altered brain development, especially in gray matter.
The importance of BRD1 in PMS goes much further than structural brain anomalies. It has been known for some time that BRD1 impacts 100s of other genes through gene regulation (epigenetics), and the role of BRD1 shifts from development in utero (fetus) to a different role after birth (Dyrvig et al 2017). Now, a new epigenetics study of people with PMS Type 1 (terminal deletions) confirms that loss of BRD1 produces widespread changes in the human genome. BRD1 is a crucial gene lost in people with deletions greater than 1 Mb.
There is little doubt at this point that BRD1 plays a crucial role in PMS.
PMS is a contiguous chromosomal deletion syndrome, meaning that larger deletions interrupt more genes of importance. BRD1 is a critical gene for brain development. If we want to understand PMS, we need detailed studies that explore more genes like BRD1. One great way to study the impact of different genes is to look more deeply at the phenotypes and genotypes of people with interstitial deletions (PMS Type 2). A treasure trove of new information awaits these studies. Not exploring these candidate genes of PMS is a waste precious time. My son, David, and so many others, are waiting for these studies. We need to do the best possible science if we are ever going to find effective treatments.
Originally posted 24 November 2018
Updated 27 November 2021
Phelan McDermid syndrome (PMS) is an intellectual disability developmental disorder. The most common reported form is a “terminal deletion” of the q end of chromosome 22. A terminal deletion occurs when a continuous segment of the chromosome is broken off at the end. Terminal deletions lead to intellectual disability (ID), language problems and coordination problems. Most people with PMS have additional problems (sleep disorder, feeding disorder, seizures, etc.), and these problems can often be severe. Many, perhaps most, have autism spectrum characteristics. I would argue that the hallmark trait of PMS is intellectual disability, because that manifestation of the disorder is the only one that occurs in 100% of people with a terminal deletion. By consensus, cases of unusual variants of the gene SHANK3 are also called PMS. These pathological variants of SHANK3 can have many of the same manifestations as seen with terminal deletions, although not all.
There are two related conditions that have lead to disagreement regarding what is and is not PMS. First, are deletions of 22q13.3 that do not disrupt SHANK3 (often called “interstitial deletions”) still called PMS? Second, does a person with a SHANK3 disruption, but without the hallmark manifestation (ID), have PMS? Out of consistency, I would argue that a 22q13.3 deletions that causes ID should be called PMS, and SHANK3 variants that do not cause ID should not be called PMS. To go one step further, given two people with the same SHANK3 variant, one person might have ID (and therefor, PMS) and another person might not have ID. This unusual combination of circumstances has been observed. Likewise, two people with the same 22q13.3 interstitial deletion might or might not have PMS. This sort of definition is common with neurodevelopmental genetic syndromes. The genetics (called “genotype”) must cause a matching set of manifestations (called “phenotype”), to meet criteria for a named disorder. Some disorders have subtypes, but PMS has no official subtypes yet.
The rest of this discussion does not depend whether you call interstitial deletions PMS, treat interstitial deletions as a future subtype of PMS, or just avoid giving them a name. Understanding interstitial deletions is crucial to understanding PMS, because PMS can include the deletion of up to 108 different genes. Some of these genes are very important. If we want to end the suffering of PMS, we need to know which genes are important and in what way. Interstitial deletions can help teach us. Let me explain how, starting with old radios.
When I was a child I took apart radios to understand how they worked. This was a dangerous undertaking for a young boy. Radios were high voltage affairs in the old days. If the power mains didn’t kill you, burned fingers from hot vacuum tubes or a hot soldering iron left painful reminders of what not to touch. My logic in those days was to remove parts until the radio stopped working. The obviously necessary part was then soldered back into place and the hunt for more nonessential parts continued. When done, I still had a working radio, plus a collection of spare parts. “Working” did not always mean “working perfectly”.
Fifty years later, teams of scientists have used this same logic to grade the importance of each gene in the human genome. One such measure is the pLI score. Think of all people who are healthy enough to have children. Analyze every gene in every one of these people. Make a note of which genes in these healthy people are missing or incomplete in some way. These are the nonessential parts. A gene that is almost never missing or incomplete gets a pLI score of 1. It must be important. A gene that is often missing or incomplete gets a pLI score of zero. You can sound technical by calling the score a measure of reproductive fitness, but the theory is no more complicated than a 10-year-old with a soldering iron. Essential parts are almost never missing from people or radios. The pLI score is the measure of gene importance.
In 2018, a team of scientists studied all deletions greater than 50 Kb in groups of people with ID (Huguet et al 2018). Basically, they asked the question, “Can ID be explained by looking at the deletion size or (similarly) counting the number of genes deleted?” They came up with a formula: add up the pLI scores of all the deleted genes, multiple by about 2.6, then add the impact of known ID genes. That gives you the number of IQ points lost due to the deletion. (Technical note: I have averaged performance IQ and verbal IQ together).
Everyone has heard of IQ to measure intellectual ability. The IQ measure was designed so the median score on an IQ test is 100 across a large population. The work of Huguet et al, including subsequent work shows that you can predict the IQ loss caused by a deletion. A deletion removing genes with a cumulative pLI of 10 will reduce a persons IQ score by about 26 IQ points. The expected IQ of a person with such a deletion would be 100-26=74. This is not a good way to predict a child’s future IQ, since we don’t know if the child’s IQ would have been 75 or 125 without a deletion. But, if the prediction is a loss of 26 IQ points and the person has mild ID, it is likely that the genetic result essentially explains that person’s intellectual disability. There is an on-line tool to help do the calculation.
The next part is a little complicated, but PMS deletions are complicated. I hope everyone can understand at least the main points.
When we apply this IQ calculation to PMS, lots of strange things about PMS start to make sense. I will use a graph to explain. The graph below shows the number of IQ points that are lost when each part of chromosome 22 is deleted. It is a prediction based on some reasonable assumptions (which will not be discussed here). Read the graph from upper left to bottom right. The graph tracks how much the IQ falls as deletion sizes get larger and larger. I have an explanation below for each numbered circle on the graph.
Circle 1: Loss of SHANK3 at the very end of the chromosome (top left corner) has a major impact on intellectual function (IQ). See how the curve drops from 0 to -30 IQ points next to circle 1? I have assumed a SHANK3 deletion costs 30 IQ points, which is a big drop even for an identified intellectual disability gene.
Circle 2: Deletion of the next 1 Mb of the chromosome has a cost of another 20 IQ points. Already, we see that deletion of SHANK3 is not necessary to reduce ID. We also see that even relatively small 22q13.3 deletions (e.g. 1 Mb) can have a large impact over-and-above the loss SHANK3.
Circle 3: See how flat the curve is at circle 3? Additional deletion of the chromosome between 1.1 Mb and 4.1 Mb has virtually no impact. For those people who say that deletion size does not matter, that is why there are so many examples. The curve is flat and, indeed, in that region increased deletion size does not influence IQ.
Circle 5: An important intellectual disability gene shows up about 8.4 Mb from the end of the chromosome. This causes another steep drop in the curve comparable to (perhaps larger than) SHANK3. See my earlier blog about this gene (TCF20).
Circle 6: I have created a hypothetical example of a 2 Mb interstitial deletion. A 2 Mb deletion is about half the size of an average 22q13.3 deletion. This deletion causes a drop in IQ (27 IQ points) that is roughly equivalent to a SHANK3 deletion. Thus, from an intellectual disability perspective, interstitial deletions can easily be equivalent to other, more common cases of PMS.
This method of studying IQ impact of chromosome deletions was not created specifically for PMS, but it seems to apply very nicely. To accurately apply this method, we need to accurately measure the IQ cost of a complete SHANK3 deletion without including the effects of other genes. Calibrating the IQ cost of deletions, including SHANK3 loss, can be done by carefully studying cases of interstitial deletions. Current data from the longitudinal PMS studies may be sufficient if more effort is put into the analysis of interstitial deletions. Finally, the method can be used to identify who might need further testing. If the estimated IQ loss does not agree with the deletion size, the person could have other genetic issues worth exploring: a mismatch could be used to justify more detailed testing (e.g., whole exome sequencing).
The research in IQ loss associated with chromosome deletion shows that, for most people with 22q13.3 deletion syndrome, fixing SHANK3 is likely to be beneficial, but not a cure. SHANK3 accounts for less than half the intellectual disability for an average size deletion (4.5 Mb) and less than 25% of a large deletion. Finally, we need to take interstitial deletions more seriously. From a scientific perspective they are hugely informative. From a PMS perspective, they are part of the same disorder.
Interstitial deletions matter because 1) they can be used to help calibrate IQ loss as a function of deletion size, 2) they can help identify which genes cause some of the manifestations of PMS not caused by SHANK3, and 3) effective treatments of PMS will depend upon how well we can treat all the important genes of 22q13.3.
Technical description: The evidence from recording of cerebellar granular cells indicate that NMDA receptors at synapses are dysfunctional in the Ib2 knockout mouse. The receptors are overstimulated, which disrupts the excitation/inhibition (E/I) balance of the neurons. Computer models show that the dysfunction of the NMDA receptors can explain the severe synaptopathy in the mouse’s cerebellum.
The practical result is poor cerebellar function. What does the cerebellum do? Wikipedia explains: “The human cerebellum…contributes to coordination, precision, and accurate timing…Cerebellar damage produces disorders in fine movement, equilibrium, posture, and motor learning in humans.” Sound familiar? To me, this sounds like David (pictured, above). He has problems learning any new motor task. He has problems retaining skills he has learned. His balance problems undoubtedly come from problems with his cerebellum.
Why is this problem so common among PMS children? That is easy to answer. Aside from SHANK3, MAPK8IP2 is the most frequently lost important gene of PMS. The two genes are very near each other on the chromosome and the vast majority of terminal deletions impact both genes.
It is possible that loss of SHANK3 contributes, to some degree, to the problem in the cerebellum. Shank3 is present in one cell type of the cerebellum. However, the new research shows that the major dysfunction produced by loss of Mapk8ip2 occurs independently of Shank3.
It is nice to start getting some answers. MAPK8IP2 is likely the most important PMS gene for balance and fine motor control. Even more exciting is that earlier work showed an already-approved FDA medication, memantine, improved behavior in Ib2 knockout mice. I wonder if this medication could help David now?
I don’t know how big David’s deletion is, but he has all the hallmarks of a PMS individual with a large deletion. His developmental delays were substantial: walking took 6 years and full oral feeding required 3 more years. He is nonverbal and even as an adult it is difficult to estimate his receptive language.
Deletion size explains some of the differences between individuals, but any given individual may be far from the “average” for a given deletion size. Is deletion size unimportant? TCF20 is a PMS gene that can help explain some of the mystery.
I have started cataloging all the different factors that influence phenotype (the features of people with a disorder). The number of factors and how the different factors interplay is rather staggering. It has been known for over a 100 years that even a relatively small number of genetic factors can produce a rather wide spectrum of phenotype characteristics. “Phenotype variability” is the term used to describe the diversity. As I progress on the cataloging of what causes phenotype variability in PMS, I will blog on various aspects and examples.
This blog is on TCF20, an important PMS gene that is lost in large (over 8.6 Mb) terminal deletions and some interstitial deletions. I mentioned that TCF20 is an important brain development gene in an earlier blog (What do we know about PMS genes?). TCF20 has all the characteristics of an important gene based on several different studies. At the time I wrote that blog I did not notice a paper (Prevalence and architecture of de novo mutations in developmental disorders) in Nature, a top scientific journal. In that study, the authors were able to affirm TCF20‘s role in genetic disorders. The cases they studied were not PMS, with large deletions. These cases were de novo mutations. Their results show that loss of TCF20 function can, on its own, cause a developmental disorder. It is yet another reminder that a number of PMS genes can cause disorders on their own, without any involvement of SHANK3.
This blog is about phenotype variability. TCF20 provides not one, but two examples of variability. These two factors operate together to explain why some kids with large deletions are more impacted by deletion size than other PMS kids.
Large deletions that are almost the same size can be very different from each other. An 8.5 Mb deletion does not impact TCF20, whereas a 8.6 Mb does. We can be confused about the impact of deletion size if we do not look closely at the genes. That is the first factor: a small change in deletion size can have a large effect. Note that the opposite can also be true. In some locations on chromosome 22, large changes (500 kb or more) can be unimportant.
The second factor is a bit more subtle. A recent paper has affirmed something else about TCF20. TCF20 is especially sensitive to “genomic imprinting” (Genome-wide survey of parent-of-origin effects on DNA methylation identifies candidate imprinted loci in humans). Normally, either copy of a gene is used by the cell. Genomic imprinting is when only one copy of a gene is used by the cell. The other copy is permanently turned off, never used. Consider this, if someone has a large deletion, but the deletion removed the copy of TCF20 already turned off, the deletion will have no effect on the production of TCF20 protein (a transcription factor). On the other hand, if the large deletion removed the active copy of TCF20, no TCF20 protein will be produced by the cell. Thus, for TCF20, “genomic imprinting” can determine whether deletions over 8.6 Mb are more devastating than smaller deletions. The two factors, deletion size and genomic imprinting, operate together. We cannot predict the effect of one without understanding the other.
Very few PMS genes are subject to genetic imprinting, but this story serves as an example. We have the scientific tools to explain phenotype variability. There are cases where deletion size seems unimportant, but these cases can be explained. The many factors that influence the future of a baby with PMS are not magical. Many people have overestimated the role of SHANK3 because PMS phenotype variably seems so mysterious. Genetics are complicated, but not mysterious. TCF20 provides a great example of how applying the science carefully can uncloak some of the mystery.
David, like most people with Phelan-McDermid syndrome (PMS), is missing one of his two SHANK3 genes. One copy of SHANK3 is gone, the other copy is intact. Some people with PMS have both copies fully intact (often called interstitial deletions) and some people have small or large disruptions of SHANK3 without affecting other genes (often called variants or mutations). For the vast majority of people with PMS, loss of SHANK3 contributes to the problems of PMS. Fixing SHANK3 has been a priority in the PMS world, although fixing SHANK3 won’t necessarily fix PMS (see Which PMS genes are most important?). I am an advocate for studying (and fixing) the other critical genes of PMS (see Looking for opportunities and Why don’t we have better drugs for 22q13 deletion syndrome?). Too much focus on SHANK3 has been an impediment to progress. Regardless, this blog is about SHANK3 research.
Science is slow
The autism world is hot on SHANK3, so research on this gene has moved forward relatively quickly. That sounds like good news, and it is, as long as we recognize that “quickly” is measured in decades. Research on SHANK3 started when my son, David, was 12 years-old. He is now 32. Over the past 20 years there have been many papers heralding the “rescue” of autism-behaviors in SHANK3 mice. Parents need to understand that “rescue” is mouse research jargon for “our genetically modified mouse is different from normal mice, and the drug we tested made them a little more like normal mice”. It does not mean “a treatment is almost here”.
The reality of research progress is less rosy than the headlines. Most research studies are done these days on rodents. A mouse with a modified SHANK3 gene is nothing like a human with PMS. Most mouse models are SHANK3 mutations, not deletions. Most people with PMS have deletions (see Gene deletion versus mutation: sometimes missing a gene is better), and human deletions affect other critical genes (see How do I know which genes are missing? and Which PMS genes are most important?). Most “SHANK3 mice” have mutations in both copies of the gene. Mutation of just one copy often results in no detectable effect. Contrast that with humans, where it is unlikely that humans can even survive without at least one working copy of SHANK3. Further, although this should be obvious, only rodent researchers talk about “autism-like behavior” in rodents. It is a rather strange concept. Measuring autism in PMS patients is difficult and sometimes controversial. There is no measure of autism in rodents, just tests to see if a mouse prefers exploring another mouse over an inanimate object. Why a mouse might do that is anyone’s guess. Certainly, you won’t have much luck asking the mouse.
It is not simply bad luck that multiple drug rescues have been reported in mice without the development of any PMS or even autism drugs that work on the core symptoms in people. The reality is that not enough is understood about the relationship among genes, drugs and behaviors. To date, there are no PMS-specific drugs currently available for testing on people. If you get invited into a drug study, that drug was invented for some other purpose. Most often, it was a drug that failed its original purpose and researchers or drug companies are looking for a different disease group to test it on.
Despite the limitations of mouse research for testing drugs, mouse research does help us learn about the proteins used in the brain and what category of drugs might someday be useful for treatment.
Twenty years of looking at SHANK3 has laid important groundwork. More recent studies have benefited from these earlier studies and from development of new research tools. Researchers are beginning to address the complexity of SHANK3 regulation. SHANK3, like most genes, is simply a recipe for making its protein, shank3 (note lower case spelling, no italics). The recipe is copied onto a template called mRNA, and then many copies of shank3 are manufactured using the mRNA template. The shank3 protein is then delivered to the synapses that need more. Like any manufactured commodity, you need to manage the supply to meet the demand. The manufacture takes place at the factory of the cell (soma) and shank3 is used in 10,000 to 100,000 or more synaptic sites elsewhere in the cell. Thus, there is an ordering and distribution network throughout the cell. Orders for more shank3 come from thousands of different sites. SHANK3 regulation is the complex process of making sure the right amount of shank3 is available at each synapse at any given time.
Synapses are the communications connection between neurons. Each synapse has a different strength of connection, and together, turn the protoplasm of the brain into an amazing computer-like machine. The machine is constantly adjusting itself to perform the tasks we call learning, memory, skill acquisition, and decision-making. Like a muscle, when a synapse is used more, it tends to get stronger and larger. That is how the computer adjusts itself. The increase and decrease of shank3 is important for these adjustments. Thus, processes like learning and memory rely on having the right amount of shank3 at the synapse. Regulation of shank3 production, distribution and utilization starts with the amount of synaptic activity at each of thousands of synapses. Complex processes connect synaptic activity with every step of shank3 production and distribution.
New research on SHANK3 regulation
There are two new papers on SHANK3 regulation that represent the next steps in understanding how the cell manages the amount of shank3 protein at each synapse. One paper forces us to rethink about what a shank3 deficiency really means.
Campbell and Sheng just published a paper on DUB enzymes and the regulation of shank3 at the synapse (USP8 Deubiquitinates SHANK3 to Control Synapse Density and SHANK3 Activity-dependent Protein Levels). DUB enzymes prevent the destruction of a protein. Normally, shank3 is destroyed after use by the USP system. These authors have identified an enzyme “USP8”. When the synapse gets very active, USP8 finds shank3 (and shank1) molecules already tagged for destruction by the USP system, and untags them. It is part of the cell’s natural system to retain extra shank3 in anticipation of needing to build up the synapse for future increases in activity. The authors point out that drugs might someday be found to mimic USP8 and help increase the amount of shank3 at the synapse of those people who have insufficient shank3 production.
In the other recent study, work by Yan and her colleagues has teased-apart some of the communications between the synapse and the nucleus used to regulate shank3 production (Social deficits in Shank3-deficient mouse models of autism are rescued by histone deacetylase (HDAC) inhibition). They show that too little shank3 at the synapse can trigger movement of β-catenin to the nucleus. The surprising part is that when β-catenin reaches the nucleus, it triggers a series of genetic effects that lead to unusual mouse social behavior. The unusual behaviors can be turned on and off independently of shank3. Thus, the strange social behaviors of these mice are not caused by shank3 levels directly. Rather, the reduced shank3 simply releases β-catenin. It is a case of synapse regulation system gone awry. These results suggest that shank3 is not the most important protein for poor social behavior. Rather, the reduced level of shank3 triggers a series of events that improperly regulate other brain genes. Curiously, this agrees with another recent study of proteins in humans with autism (see Which PMS genes are most associated with Autism?).
This is the end of the blog except for a few final thoughts, below. For those who wish to dig a little deeper into the science, I have provided additional background material on genes, proteins and regulation called “A primer on the lifespan of a protein“.
A primer on the lifespan of a protein
This is a primer about the lifespan of proteins. Shank3 is a protein (note lower case spelling for the protein). Like nearly all proteins, it is born, gets called-upon to work, does its job, then is dissolved and discarded. The most important point is, the amount of shank3 in the cell is highly regulated. At any given moment, there are complicated processes deciding how much is needed and where it is needed. Usually, some parts of a cell may be building up shank3 supplies and incorporating them into synapses, while other parts of the cell are breaking down shank scaffolding (structures built from shank molecules). I sometimes think of shank3 as a construction material, like plywood. Lots of wood scaffolding may be used to frame a concrete pillar. After the pillar is in place, the wood may be torn down and discarded. Some wood may be discarded at the same time other is being nailed in place for the next pillar, wall or sidewalk. You always want a supply of wood around, but that supply should never be more than the anticipated need. Construction management involves reading blueprints, anticipating needs, ordering the manufacture of materials, and delivering what is needed, on time. Timely and organized manufacture, distribution, utilization and disposal of shank3 very important for the cell.
The SHANK3 gene is simply a blueprint for shank3 protein. The blueprint is converted into a template for stamping out the protein (call mRNA). Gene regulation (via “promoters”) control how many copies of mRNA are created. Each mRNA is degraded and discarded after a certain number of copies of shank3 have been made. While functioning, mRNA is used to stamp out copies of shank3. Shank3 is then transported and collected in a pool of proteins near the synapse. The synapse is a very active site, like the beehive of activity at a busy construction site. Shank3 molecules near the synapse gets incorporated into the “post-synaptic density” as needed. The buzz of activity includes constantly building up and breaking down of shank3 molecules. Old shank3, whether it be after a piece of scaffolding is no longer needed, or if the molecule has been sitting around unused, is degraded and discarded.
Breakdown and disposal of shank3 protein
Shank3 is tagged and trashed by the USP system (ubiquitin-proteasome system). Ubiquitin tags it and the proteasome dissolves it. That is the last step in the life cycle. There is a way of untagging, with a deubiquitinating enzyme (DUB), which I would never even mention except that a recent study looks at untagging as a way to increase shank3 levels at the synapse.
Regulation of shank3 occurs at every step
Many different processes regulate transcription (making the mRNA template), synthesis (stamping out shank3 molecules), incorporation (using the shank3), and degradation (USP system). Each of these processes is a potential therapeutic target for increasing shank3 protein in people who have only one working copy of the SHANK3 gene.
Early work on shank proteins (shank1, shank2 and shank3) focused on how each protein is used at the synapse. Increases in shanks are associated with establishing and maintaining strong synaptic connections, whereas decreases can inhibit the formation of new connections. Too much or too little shank3 affects the sizes or number of synapses. Synapse size is related to information transmission in the brain. Too much information from the wrong channel creates noise. Too little information interferes with learning, memory, skill acquisition or other function. Thus, the early research looked closely at shank3 levels at the synapse.
Shank3 production is largely regulated in the cell nucleus where DNA is found. Each brain cell has only one nucleus, yet it regulates shank3 production for thousands to over 100,000 synapses in that cell. So, the signal to increase and decrease Shank3 production comes from a potentially huge number of synapses and converges at the one and only nucleus. Transcription (making the mRNA template) is regulated by many mechanisms. Most mechanisms influence the “promoter” region of a gene. That is the region where the hardware for reading DNA and making the mRNA template is assembled to do that task. A dizzying array of molecules influence the promoter region. To complicate things even more, shank3 has not one, but 7 total promoter regions that regulate not only when to transcribe, but also which of the 20 to 100 various versions of Shank3 to produce. Yes, there are at minimum 20 versions (isoforms) of shank3 produced during a person’s lifetime. “Turning on and off” a gene is another way of saying the gene is set to transcribe or not. In actuality, neurons that use shank3 don’t turn the genes on or off. Rather, they increase and decrease transcription rate of one, two, up to 20 isoforms of shank3. If it sounds complex, it is. Most papers focus on one or two isoforms. They overlook the rest to make the research manageable. For the moment, it is enough that we recognize that transcription of the SHANK3 gene for making mRNA and many copies of the shank3 protein is regulated at each step. The gene is regulated largely at the promoters, controlling how many mRNA molecules are created for which isoforms. Recent studies have looked for ways to modify the regulation of transcription to compensate for a missing copy of the SHANK3 gene.
Shank3 mRNA is used to synthesize shank3 protein. How rapidly shank3 protein is produced is, like the other steps, under careful regulation. For example, each mRNA does not last forever. At some point it is disassembled (degraded) and can no longer produce protein. Its regulation is yet another possible way to manage shank3 protein production.
As mentioned earlier, shank3 protein is used (or just sits around waiting), and is then broken down by the USP system. Unlike signaling in the nucleus, the USP system is operating at or near the synapse. It can influence shank3 availability on a fine grained scale.
Increasing or decreasing shank3 with blunt instruments
Each step along the life cycle of shank3 is an opportunity to increase the amount of shank3 in the cell. One might be eager to try one or many of the steps on mice or people. This eagerness should be tempered by the potential pitfalls of circumventing the normal regulatory process of the cell. Let’s remember why shank3 is so highly regulated. Shank3 levels must be adjusted at each synapse on an individual basis. Large cells can have up to 200,000 synapses. If we choose to increase shank3 transcription in the nucleus, we may start to force some, perhaps too many, synapses to overproduce shank3. The cell needs to remove unnecessary and unwanted synapses (called “pruning”). Pruning is one of the most important processes in brain development. Another related process is called synaptic homeostasis. One theory about why sleep is important is that it gives the brain a chance to readjust all the synapses to consolidate learning and properly reset the brain for new learning the following day. Both pruning and homeostasis are likely affected by changes in overall shank3 levels.
A drug that simply increases the shank3 in a cell could be beneficial, but we must be wary of blunt instruments. We must be concerned that increasing shank3 by short-cutting the built-in regulation of shank3 may worsen PMS, or perhaps replacing one problem with a new one. This is why a potential drug treatment requires thorough testing in animals to develop a deep understanding of the mechanism of action. Improving one behavior in a mouse is not enough. At a minimum, we need to carefully explore what other behaviors might be affected in the model animal. In this blog, I have not discussed that SHANK3 is used in different ways in different parts of the brain, and regulation is likely to vary for each brain region. Methods to deliver different amounts of a drug to different brain regions are complex and experimental.
Mice are handy experimental animals, but their brain function is not at all like human brain function. In mice, you can remove 100% of all shank3 protein and the mice, although behaviorally unusual, are able to eat, drink, run, play and procreate much like normal mice. Humans missing both copies of SHANK3 are unheard of, most likely because they don’t survive in the womb.
The take-home messages from the new research are simple enough. First, there is much hype in the online press reports and magazines about scientific progress. The fact is, science is slow and we have a long way to go. The details are sometimes complicated, but the basic principles are not. Second, studies of shank3 have not reached the point where we truly understand the relationship between SHANK3 gene loss and the many problems that result. Progress is being made, but, as often happens in science, the latest results tell us what we thought we understood was not exactly correct.
What the new study shows is, regardless how a person gets autism or schizophrenia, the same networks of genes become dysregulated. Let’s first discuss what gene regulation means. DNA is like a well-stocked bakery. A good cook can prepare many different kinds of breads or desserts by choosing how much of each ingredient to use, and when. Just about every cell in the body has the same DNA. What makes one part of the body different from another is how much, and when, each gene is used. DNA cooking is called gene regulation. In autism and schizophrenia, the proportions of ingredients have gone awry.
The green diagram at the top of this blog maps the results of the new study. The researchers found certain critical “modules” (functional groups) of genes that are dysregulated in the brains of individuals with these two disorders. Once, again, these genes are dysregulated regardless of how one acquires autism or schizophrenia. The map identifies the 20 most dysregulated genes in each module (140 total) and how they interact in the brain.
What does this diagram tell us? It says some things we already knew. Autism (and schizophrenia) cause problems in neurons, the brain cells responsible for sensation, thinking and action. Less obvious, autism seems to be related to two other cell types, astrocytes and microglia. Astrocytes nourish neurons. Microglia, which also come in contact with neurons, are known to regulate the formation and removal of synapses. There are other important cell types, as well.
What is the news for PMS? We learn that two PMS genes are core genes of the dysregulated neuron networks. I have circled these genes in RED. There are about 20,000 genes in the human genome. The paper identifies the top 140 dysregulated genes. Obviously, they are quite important for psychiatric disorders. The two PMS genes are MAPK8IP2 and SULT4A1. Not surprisingly, MAPK8IP2 and SULT4A1 have already been identified as two of the 18 most important genes of PMS (see Which PMS genes are most important?).
Which individuals with PMS are missing these genes? Nearly all (over 95%) of people with PMS are missing MAPK8IP2. About 30% of people with PMS are missing both MAPK8IP2 and SULT4A1. If your child has a typical (terminal) deletion, you can look up which important PMS genes are missing in this blog: Which PMS genes are most important?
At this point, it seems pretty likely that deletions of 22q13.3 do more than raise the risk of autism. Deletions can directly impact MAPK8IP2 and SULT4A1, two core genes dysregulated in autism, schizophrenia and other neuropsychiatric disorders. Perhaps the good news is that people who study autism and schizophrenia have a new impetus to study MAPK8IP2 and SULT4A1. It is up to PMS parents to lobby, cajole and otherwise let everyone know that studying these genes is very important to us.
Phelan McDermid syndrome (22q13 deletion syndrome or PMS) is often equated with autism spectrum disorder (ASD). The exact definition of PMS is somewhat murky (see Defining Phelan McDermid syndrome). There are disagreements among families, scientists and clinicians. The controversies have been around for at least 9 years and remain a sticking point for parents trying to get diagnoses and services for their child. Equally messy, it seems, is the relationship between PMS and ASD. Some studies report that up to 70% of their PMS patient population have an ASD diagnosis; others find as low as 30%. Many parents admit they have received a somewhat arbitrary ASD diagnosis from clinicians to help their child receive services. One scientific study that looked closely at the symptoms of PMS patients argued the behaviors are not really ASD. Another study showed the ASD diagnosis is unreliable in children with both intellectual disabilities and movement problems. Two studies suggested the number of cases with ASD depends on the sizes of the chromosomal deletion in the population. No wonder there is so much confusion regarding the incidence of ASD among PMS patients.
There is a misconception among many parents that a case of PMS that involves the SHANK3 gene must lead to ASD, since “SHANK3 is an autism gene”. For the record, there are no “autism genes”, only autism-associated genes. The SFARI organization tracks genes that are associated with ASD in their SFARI Gene database. There are currently about 800 autism-associated genes categorized in the database. SHANK3 is a category 1 gene, meaning that the association with autism is high. What does that mean?
Most genes of the human genome come in slightly different flavors. Each flavor is called a “variant”. The SFARI database tracks rare variants that can strongly contribute to autism. That is, the most common versions of the SHANK3 found in the general population does its job without any problem. What sometimes happens in PMS is that the parents have common versions of SHANK3, but the child has a copy of SHANK3 that is significantly different from her parents. If the child has a resulting disorder, the changed gene is called a “deleterious de novo” event. In this context, deleterious means damaging and de novo means new, since the parents have the more common variants of the gene. A deleterious denovo event might be a chromosome deletion (like 22q13 deletion) or a gene mutation (as in our SHANK3 example). It is no mystery why rare deleterious gene variants are rare. They are rare because people with a serious genetic problem do not tend to have children of their own.
The SFARI gene database is mostly concerned with rare variants. However, common variants can also contribute to a disorder. In fact, in ASD most cases are a result of common variants. Let me explain how common variants and rare variants contribute to a disorder using a metaphor. Let’s say you are on a whale-watching boat filled with lots of people. Today is unusual, the normally calm waters have large waves. This is a rare event. On choppy seas the waves in the water can make the boat rock back and forth. The water is rough and that is associated with a risk of capsizing. With a seaworthy vessel the passengers are in no immediate danger. Of course, a single person walking from one side of the boat to the other side has no noticeable influence on the boat. However, if too many people move to one side of the boat a large wave might capsize the boat and send everyone into the water. This is a combination of a high risk rare event (choppy seas) and many small contributions all in one direction (too many people on one side). The combination can lead to disaster.
Like the people on the boat, most variants are quite common and contribute only a tiny bit on their own. These variants are very common in people. But, if you have too many common variants on the autism side of the boat, you have a major risk of developing autism. An autism-associated gene is rare and can strongly contribute to autism. Like choppy seas, a single autism-associated gene can greatly raise the risk of autism. The combination of a deleterious variant of an autism-associated gene, plus enough common variants in one direction (towards autism), can pass the tipping point and produce a case of autism.
To be clear, everyone has variants in their genome. Too many variants of genes can raise the risk of many disorders, from diabetes to heart disease. Judging the risk of a disorder from a person’s common variants is called polygenic risk and is measured using polygenic risk scores.
What about PMS? PMS occurs primarily by a partial (“terminal”) deletion of chromosome 22. That deletion often includes SHANK3, BRD1, CELSR1, and SULT4A1, each associated with intellectual disability or other neurodevelopmental disorder. SHANK3 is most commonly affected because it sits near the end of the chromosome where breaks occur most often. In addition, SHANK3 is a large gene that can have a substantial impact if disrupted by a deleterious de novo event.
I began by explaining there are two types of genes that can contribute to autism. There are common variants that, together, can add to the risk of autism. The other type of gene variant, one that may arise from a deleterious de novo event, is rare and makes a large contribution to autism risk. Some SHANK3 variants are common and occur throughout the population. Some rare variants greatly raise the risk of autism. In PMS, it is the combined risk of too many common variants on one side of the ship, plus a deleterious de novo event (usually a deletion), that can send a child tumbling into ASD.
In general, most people with autism (about 70%) do not have a rare variant of an autism-associated gene. Their autism results from many common variants that have combined with developmental and environmental factors to produce autism. In a child with both PMS and ASD, the combined impact of common variants plus a de novo events (chromosomal deletion or pathogenic SHANK3 variant) leads to ASD. In other children with PMS, the genetic change on chromosome 22 combined with the common variants of the child just don’t add up to ASD. It is likely that in some cases of PMS, the the common variants in a child’s genome strongly reduce the likelihood of ASD and thus have a protective effect.
Two very recent studies of Phelan McDermid syndrome (PMS) drew exactly the same conclusion: We need to recruit and study more PMS patients with interstitial deletions if we are going to understand the syndrome (see references 1 and 2, below). This blog explains why that is a critical need. In some ways, this blog is an update to an earlier blog (Why don’t we have better drugs for 22q13 deletion syndrome?).
PMS can be broken down into a few obvious classes. The original disorder, 22q13.3 deletion syndrome, has terminal deletions and interstitial deletions. Later, SHANK3 variants (often called “mutations”) were added. As I have discussed before (Gene deletion versus mutation: sometimes missing a gene is better), mutations are a mixed bag. Some mutations produce symptoms like 22q13.3 deletion syndrome, but other mutations produce other disorders (like ASD or Aspergers), or no disorder at all.
PMS research started out with SHANK3, but somehow it got stuck there. Being stuck has led to some serious deficiencies in our understanding of PMS. First, very little is being done for the future of children with interstitial deletions. Their SHANK3 gene is intact, so SHANK3 research does them no good. Second, drug studies that use PMS patients to study SHANK3 are likely to fail without accounting for the important genes in each PMS patient. This was discussed in the recent paper on PMS genes (reference 2). PMS patients have such a mix of deleted genes that the benefits of a drug for SHANK3 loss might not be detectable. Third, certain serious problems seen in PMS are unlikely a result of SHANK3. These issues, like poor thermoregulation (body temperature control), lymphedema, cerebellar malformation, mitochondrial problems, and certain developmental problems, impact a large proportion of children with PMS. Every year children and adults with PMS die. We need to know which genes are associated with lethality. These issues will remain serious problems for people with PMS as long as SHANK3 remains the narrow focus of PMS research. Even our understanding of SHANK3, itself, is incomplete without a much better understanding of the other important genes of PMS.
The best way to understand the many genes of PMS is to study people with interstitial deletions. They are the only PMS patients where we can safely say that SHANK3 deletion does not play a role. My last two blogs show that we actually know a lot about PMS genes that are most likely to cause problems. However, we need to know much more about how each of these genes affect people. That requires people with different size interstitial deletions.
There was one research study of people with interstitial deletions published in 2014 (Disciglio et al.). It covered 12 patients. Since that paper, there has been only one additional (single) case study of an interstitial deletion. By comparison, PubMed shows 164 papers with SHANK3 in the title. Most PMS families are probably not aware that the current major studies of PMS specifically exclude interstitial patients: Natural History of Phelan McDermid Syndrome and the Electrophysiological Biomarkers of Phelan-McDermid Syndrome. Some of the sites in these multisite studies have not excluded participants with interstitial deletions, recognizing the scientific importance of these cases. Scientifically, excluding interstitial deletion patients makes no sense. We should be seeking them out, recruiting them. As a parent, excluding interstitial deletions seems unfair to both those families, and to the rest of us. We need to get unstuck. We need the best science possible to help our children.
In the previous blog we learned which Phelan McDermid syndrome (PMS) genes are most important. SHANK3 has often been touted as the gene that causes PMS, but SHANK3 rarely operates on its own and in some people has nothing to do with PMS (those with interstitial deletions). We learned that large studies of human populations identify 18 PMS genes that are impacted by “natural selection”. Loss of these genes are highly likely to cause problems, the problems that add up to PMS. The genes are:
The big question is, what do we know about these genes? That is, how might they be contributing to PMS? This blog is based on a paper that not only identified these genes, but also pulled together what is currently known about each. Although the paper describes each gene’s function in detail (for well-characterized genes), it also classifies genes into groups. Those groups are quite informative and help us understand why PMS has certain characteristics.
Genes that impact brain development
It is now very clear why PMS can occur with or without SHANK3. Of the 18 PMS genes that are likely to have a high impact on PMS, at least 7 impact brain development: SHANK3, MAPK8IP2, PLXNB2, BRD1, CELSR1, SULT4A1, TCF20.
PLXNB2 regulates the growth of neurons, especially early in development. PMS is a neurodevelopmental disorder, so nothing could be more important than regulating neuron growth.
CELSR1 is also crucial for neurodevelopment. Neurons are exquisitely organized into nuclei in the deep structures of the brain and into very precise layering in the cortex. For example, pyramidal neurons of the cortex are located only in certain layers of the cortex, with the dendrites reaching upwards and the axon pointing down. The axon often winds its way towards the white matter. CELSR1 is important for orchestrating the orientation of neurons to assure proper organization.
BRD1 regulates hundreds of other genes during development. It is highly associated with schizophrenia, as well as PMS. PMS individuals with terminal deletions greater than 1 Mb are missing BRD1 and the loss of BRD1 impacts the entire genome (see https://pubmed.ncbi.nlm.nih.gov/33407854/).
SULT4A1 was recently shown to impact the mitochondria in the brain (see New science: SULT4A1, oxidative stress and mitochondria disorder). Mitochondria convert food into energy for cells. Dysfunction of mitochondria explains why deletions that disrupt SULT4A1 can have a severe impact on neurodevelopment and adult brain function.
There are three genes that have close association with sleep or sleep disturbance. SHANK3 impacts sleep in some individuals with PMS, but PIM3 (see this paper) and PRR5 (see this paper) have been identified in studies that explore which genes regulate circadian rhythms (so called, “clock” genes).
Gene associated with lymphedema
CELSR1, the gene important for proper orientation of cells during neurodevelopment, is also associated with inherited lymphedema. Presumably CELSR1 influences cell orientation and the structure in the lymph system during development.
Genes that have unknown function
We must recognize that just because a gene has never been closely studied, that does not mean it is unimportant. In fact, one genomic study makes a convincing argument that genes of unknown function are just as important as the well-characterized genes. PMS has 7 genes likely to be important, yet not well-studied: TRABD, ZBED4, SMC1B, PHF21B, SCUBE1, SREBF2, and XRCC6. The first two genes, TRABD and ZBED4, are of very special concern. One copy of each gene is missing in over 95% of individuals with terminal deletions. It is imperative we find out what these genes are doing and how loss impacts PMS.
This study of PMS genes was a critical step forward in understanding PMS. It provided a short list of culprits. It explains why interstitial deletions cause PMS and it identifies where our research efforts need to be focused. Most importantly, it provides new targets for therapeutics. Unfortunately, a lot of time has gone by without any serious effort to encourage research into the full array of PMS genes. The genes listed above warrant much more study. As a parent of a child with PMS, I strongly feel we should make an effort to encourage the scientists who study these genes. It is hard to understand why so many important genes of PMS are being ignored by the PMS community.
If you ask someone familiar with 22q13 deletion syndrome (Phelan McDermid Syndrome, PMS), “which genes are most important?”, they will start with SHANK3. Even though some people with PMS have no problem with SHANK3, it is still the most important gene for two reasons. First, deletions of SHANK3 and many uncommon variants of SHANK3 can have a strong negative impact on individuals. Second, a large percentage of the PMS population are missing SHANK3. Thus, SHANK3 meets the criteria of 1) potentially large impact on an individual and 2) a large percentage of the population is missing SHANK3. This blog is a closer look at all the genes that meet these two criteria.
In a recent study, a group of researchers looked at genes that are highly likely to contribute to PMS and are missing in most people with PMS (Identification of 22q13 genes most likely to contribute to Phelan McDermid syndrome [full disclosure: this blog was written by an author on the paper]). That is, genes that appear to meet the two criteria listed above. What makes this study important is that it does not differentiate between genes that have been carefully studied and genes that have never been studied. From my perspective as a parent of a child with PMS, we parents are not interested in gene popularity contests. We are interested in learning what is making our children sick.
I read that SHANK3 was the only important gene
Until recently, nearly all PMS research was focused on one well-known gene, SHANK3. Some scientists have promoted the false narrative that only the SHANK3 matters. But, PMS is a polygenetic disorder. That is, many genes are involved. For individuals who have a pathogenic variant of SHANK3 and no other genetic issues (PMS Type 3), it is true that only SHANK3 matters. But, the vast majority of people identified with PMS have chromosomal deletions (PMS Type 1). Some 97% of people identified with PMS are missing up to 108 genes! (See Understanding deletion size and How do I know which genes are missing?). Consider, as well, that some people have PMS even with intact SHANK3 (PMS Type 2). SHANK3 is clearly not the only important gene. One major goal of PMS research it to identify which genes are most important.
How can we find out which genes are most important?
There are 108 PMS genes and only 44 have been well studied. If there was no independent way to identify the important genes, we would be in serious trouble. Fortunately, there is a way to predict the importance of a gene without knowing what the gene does. A large group of scientist compiled a database of over 140,000 genomes including those of “normal” individuals (Genome Aggregation Database). Normal in this case means no developmental or neurological disorder. This huge database lets you predict which genes can cause trouble. Let me explain.
There is a trick to finding a likely troublemaker gene. Pick a gene. Look at all the copies of that gene in the database. (Each human has 2 copies of each gene.) The same genes in different people are not always the same. Different people can have slightly different versions, called “variants”. It is possible to estimate (with biology and statistics) how many different variants you would expect to occur by chance in a population. For example, for a given size gene you might expect to discover 40 different variants among 60,000 people. What if you find only 5 variants? Something’s fishy if you find only 5. The best explanation is — here is the trick — that the other 35 possible variants of that gene cause serious problems. For one reason or another, those 35 variants remove a person from the category of “normal individuals”. There is a name for this: natural selection. It is a principle discovered by Darwin and it works for genes.
Those missing 35 variants are pathogenic. The variation causes a loss of normal function, and the body cannot operate normally without the gene. Genes that are highly sensitive to variation are called “loss-of-function (LoF) intolerant”. Genes that are LoF intolerant in this way are the ones most likely to cause major health problems. Only a minority of genes are LoF intolerant. Most genes tolerate all kinds of variation. Either they are less important genes, or the body has found ways to work around variations.
Wow! Which genes are most important?
So, which PMS genes are very LoF intolerant? That is an easy question to answer. You can go to the gnomAD browser and look up any gene. Look for the row with pLoF and get the “pLI” value. A value between 0.9 and 1.0 is a bad news gene. SHANK3 is 1.0 — no surprise there, but what about other genes? Let me save you some time. Below is a list of PMS genes that have a pLI value above 0.9.
Genes in this list are in the order of their position on the chromosome. The ones at the top of the list are more frequently lost in the population. If your child has a terminal deletion, look at all the genes with a Kb value smaller than your child’s deletion size. Those are the genes that most likely contribute to his/her disorder.
The first thing to notice is that what started out as 108 genes is now reduced to 18 genes. There are a few other genes with pLI below 0.9, but not far below 0.9. These may also be important. Regardless, the number of PMS genes has gone from intractable (108) to something much more manageable (18 or maybe 20). If your child has an average size deletion (around 4,500 kb), then there are 10 relevant genes. Note that some children are missing only SHANK3. We will not forget them. But, in the meantime we need a major push to fully understand the other important genes of PMS.
In future blogs I will discuss what some of these genes do and how they might impact your child.