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. There are disagreements among families, scientists and clinicians. The controversies have been around for at least 6 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 find up to 70% of their PMS patient population has ASD; 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 990 autism-associated genes. SHANK3 is one of the 990 autism associated genes. What does that mean?
There are two types of autism-associated genes. Let me explain them by example. Let’s say you have a large boat filled with lots of people on choppy seas. The waves in the water can make the boat rock back and forth, but they pose no risk to capsizing the boat. A single person walking from one side of the boat to the other side has no visible impact on the boat in the water. Yet, send too many people to one side of the boat, then even a modest wave might capsize the boat and send everyone into the water.
Most genes of the human genome come in slightly different flavors. Each flavor is a “variant”. The SFARI database tracks those variants that can contribute to autism. Like the people on the boat, each variant contributes only a tiny bit on its own. But, if you have too many variants on the autism side of the boat, you have a major risk of developing ASD. To be clear, everyone has these variants in their genome. Only some people have enough to be at risk for autism.
What about PMS? PMS occurs primarily by a deletion on chromosome 22. That deletion often includes SHANK3, MAPK8IP2, BRD1, CELSR1, and SULT4A1, each associated with neurodevelopmental disorders. SHANK3 deletion is the most common simply because it sits near the end of the chromosome. MAPK8IP2 is almost as common because it is adjacent to SHANK3. In 90% of PMS cases they get knocked off the chromosome together. Most, if not all, of these genes cause intellectual disability when a copy is missing (haploinsufficiency). When the deletion or disruption does not exist in the parents, but does exist in the person with the disorder, it is called a “deleterious de novo” event. In this context, deleterious means damaging and de novo means new, since the parents are not missing the gene. The deleterious denovo event might be a chromosome deletion (22q13 deletion) or a gene mutation.
I began by explaining there are two types of autism-associated genes. There are common variants that, together, can add up to a huge risk of ASD, like too many passengers on one side of the boat. The other type of gene, those that arise from a deleterious de novo event, are like the large ocean waves. In and of itself, a large wave is not going to capsize the ship (cause ASD). But, the combined risk of too many common variants on one side of the ship, plus a deleterious de novo event, can send a child tumbling into ASD. This is why most people with ASD do not have a syndrome like PMS. They have many common autism-associated variants that have combined with developmental and environmental factors to produce autism. The combined impact of common variants and de novo events also explains why many children with PMS have autism or ASD-like behaviors. Perhaps they don’t have a huge overload of common variants, but they have enough when combined with the loss of PMS genes. It is also why many children with PMS are quite social, with no evidence of autism.
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.
MAPK8IP2 sits almost adjacent to SHANK3 and we have known for years that loss of MAPK8IP2 in mice interferes with brain function. 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, often winding its way towards the white matter. CELSR1 is important for orchestrating the orientation of individual neurons.
BRD1 regulates hundreds of other genes during development. It is highly associated with schizophrenia, as well as PMS.
Genes associated with sleep
There are three genes that have close association with sleep or sleep disturbance. SHANK3 impacts sleep in some individuals with PMS, but PIM3 and PRR5 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 has provided a convincing argument that genes of unknown function are 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 why their loss might be highly impactful in PMS.
This new study of PMS genes has breathed new life into PMS research. It has 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, we have new targets for therapeutics. New targets mean new hope.
Unfortunately, a lot of time has gone by without any serious effort to encourage research into the full array of PMS genes. We have not taken any advantage of 17 opportunities to make PMS children better. As a parent of a child with PMS, I strongly feel there should be greater respect for parents’ hopes. These hopes deserves every effort to search for treatment options.
Ask anyone who has read about 22q13.3 deletion syndrome (Phelan McDermid Syndrome) which genes are most important and they will start with SHANK3, even though some people who have 22q13.3 deletion syndrome are not missing SHANK3. SHANK3 is most important for two reasons. First, mutations or deletions of SHANK3 can (although not always) 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 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 same two criteria as SHANK3 for importance. 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. 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
Up until now, nearly all PMS research has been focused on one well-known gene, SHANK3. But, for the overwhelming majority of PMS sufferers, some 97% (see Understanding deletion size and How do I know which genes are missing), PMS is a polygenetic disorder. That is, many genes are involved. Is it possible, as a few SHANK3 scientists have suggested, that only the SHANK3 matters? Considering that people can have all the problems of PMS even with intact SHANK3 (called “interstitial deletions”), it does not seem possible that SHANK3 is the only gene that matters. (For the minority of parents whose child only has a SHANK3 variant or loss, SHANK3 is the only important PMS gene, but that strikes me as a rather selfish viewpoint.)
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 way to identify the important genes, we would be in serious trouble. Fortunately, the recent study of PMS genes was possible because of a recently compiled database of over 60,000 genomes of normal individuals (Exome Aggregation Consortium). Normal in this case means no developmental or neurological disorder. Why normal individuals? Because, this huge database lets you predict which genes can cause trouble.
Here is the trick to finding a likely gene troublemaker. Pick a gene. Look at 120,000 copies of that gene. (Each human has 2 copies, so 60,000 people = 120,000 copies of the gene.) Like anything else, some will be a little different than the others. In fact, you can estimate how many variants you would expect to occur by chance in a population of 60,000 normal people. So, for a given size gene, maybe you would expect 40 different variants of that gene in the population. 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 removed the owners of that gene from the population of “normal individuals”. Those missing 35 variants are pathological. They cause a loss-of-function. The gene is called loss-of-function (LoF) intolerant, and those genes that are very LoF intolerant are the ones most likely to cause major health problems.
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 EaAC web site and look up any gene. Look for the row with LoF 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 to something much more manageable. If your child has an average size deletion (around 4,500 kb), then there are 10 relevant genes. Note that some, although relatively few, children are missing only SHANK3.
In my next blog I will discuss what these genes do and how they might impact your child.