Success is very much about seizing opportunities. With all of David’s early issues, we could not address everything at once, but we always looked for opportunities. For example, when he started climbing in the refrigerator we encouraged him (under watchful eyes). See the picture here: Gene deletion versus mutation.
Science is about hard work, but it is also about seizing opportunities. The discovery of penicillin is a classic example. Alexander Fleming made his discovery in a moldy petri dish. The open dish was contaminated by a mold that killed bacteria in the dish. The mold in the dish was accidental, but Fleming’s observation was not. He was a scientist looking for ways to kill bacteria. A few years after the initial discovery, penicillin saved its first life: a child. We need to keep our eyes open for opportunities and we need to make opportunities happen. So how can we do that?
This past week a group of 22q13 deletion syndrome parents took on a challenge. I asked them to identify other children who are most like their own. The goal was to find ways to “cluster” the characteristics of children with 22q13 deletion syndrome, as described in my most recent blog: Splitting, Lumping and Clustering. It was a lot of fun and, just as I suspected, there are groups of kids that are very similar to each other. The information on the Facebook group could be compiled and studied. I would recommend someone do that. The exercise could be expanded. There is a lot to learn. Parents have insights into their children that medical researchers cannot. Categorizing how groups of children are alike and different could speed up research.
This blog is about other, untapped opportunities to look at categories of 22q13 deletion syndrome children. There are special cases we should not overlook.
I hear people say that no two deletions are exactly alike. Not true. There are cases where the deletions are exactly the same. Here is the list: 1) twins (yes, there are twins in our community), 2) unbalanced translocations (my son’s deletion and my niece’s deletion are exactly the same, as are several other children and adults in our extended family), and 3) germ line deletions. I do not know any 22q13 deletion syndrome families with multiple children from germline deletions. I would be interested in hearing of any cases.
I have heard some doctors and scientists say “no two deletions are alike” even though they should know better. We need to exploit these cases to find out what matched deletions have in common and how they differ from each other. Those observations will hint at which aspects are genetic and which are probably not.
There are a lot of 22q13 deletion syndrome children with terminal deletions. There are fewer people with interstitial deletions. What if we take each person with an interstitial deletion and matched them up with someone who’s deletion starts at the same spot on the chromosome? In such a case both people would be missing the same interstitial genes. What can we learn? It is a kind of A minus B experiment. It might tell us a lot about what the genes in common are doing.
Pure SHANK3 deletions
One popular theory about 22q13 deletion syndrome is that SHANK3 mutations act simply by reducing the amount of SHANK3 protein. If a SHANK3 gene is missing altogether, there is no controversy. However, there is an alternative theory that mutations of SHANK3 cause the formation of damaging proteins. The difference is important. In the first case, studying SHANK3 mutations are likely to help anyone with a SHANK3 loss (most people with 22q13 deletion syndrome). In the second case, a cure for SHANK3 mutation is not likely to help most people with 22q13 deletion syndrome. Right now the differences are being studied in mice and rats. As valuable as such research might be, it does not resolve the question in people.
We need a study that specifically compares these two groups, people with SHANK3 mutations and people with complete (or nearly complete) SHANK3 deletions that are small enough to leave other, nearby genes, alone. Once again, we as parents can look at our children, and start listing their characteristics and share the similarities and differences.
I believe parents can be major contributors just by our ability to see similarities and differences in our children. The scientists and clinicians studying our children have all kinds of ideas, but frankly they can use a little guidance. Drug studies are mixing kids with the tiniest mutations and kids with big deletions. Tools to measure vocalizations are being tested on kids that make very few sounds, and their parents already know what those sounds mean and how often their kids make them. We can appreciate that our kids are difficult to understand, but the whole research/investigation process can be improved. The PMSF International Registry has been a big step in the right direction, but listening to the parents explore like-children this week on Facebook, it is clear families are ready to do more.
It is very hard to talk about and explore, much less cure, a syndrome if you don’t define it first. While 22q13 deletion syndrome seems like it should be straightforward — a deletion of 22q13 — life is rarely that simple. In 2012 I was offered a chance to bring people together to address the question of how to define Phelan McDermid syndrome (PMS). I took the role and opportunity seriously. I decided to make a slide presentation that would set the stage for parents, scientists and clinicians to discuss a definition for the syndrome. As it turned out, the offer was rescinded. Without any modification, I present the final slide from my talk, 4.5 years later. In my opinion, the discussion is overdue.
The color coding is important. Things in green are PMS. Things in rust red are not PMS. Dashed lines are just to make it easier to see. The scheme covers nearly every circumstance, including pathology of regulatory sites. The only unaddressed issue is what might be considered phenotypic. It seems to me now that any intellectual disability that is not syndromic in some other way (e.g., metachromatic leukodystrophy caused by the deficiency of arylsulfatase A, OMIM #250100), should be considered the core phenotypic trait of PMS. Regardless, the slide represents the only detailed framework I have ever seen for a definition of PMS.
There is a great interest in SHANK3 and its relationship with 22q13 deletion syndrome. Using the scheme, above, and other information that we know about SHANK3 and 22q13 chromosomal deletions, I recently put together this chart:
In this case, the dashed line indicates that autism spectrum disorder may accompany intellectual disability and still be part of PMS. The chart shows that many SHANK3 mutations are not PMS. They are either nothing (have no phenotype) or some other neuropsychiatric disorder. When 22q13 deletions include SHANK3 (even just a part of SHANK3), they can be PMS. In fact, they are rarely not PMS. Some SHANK3mutations lead to the phenotypic traits of PMS. Mutations of SHANK3 that confer a different primary phenotype (e.g., schizophrenia or autism spectrum) should not be lumped into the PMS category.
There are other ways to define a disorder, but the worse thing we can do is not define it at all.
David does not talk, although I am certain he would like to. He has poor hand control. He can just barely manage a spoon or glass of water with great effort. Although he walks a lot, he is always at risk of falling. There are so many things that are difficult for David. It would be nice if we had a medication to make his life easier.
After years of drug testing on children with 22q13 deletion syndrome we are probably no closer to a treatment now than when it started. This problem is not unique to 22q13 deletion syndrome; it is true for many, if not most neuropsychiatric disorders (see: Hope for autism treatment dims as more drug trials fail). Recently, Rachel Zamzow wrote a very readable review about why autism clinical trials have failed (Why don’t we have better drugs for autism?). Her review is in Spectrum, the on-line magazine affiliated with the Simons Foundation Autism Research Initiative (SFARI). Rachel identifies three problems that plague clinical trials: 1) bad design, 2) wrong measures and 3) too broad a range of participants. While problems 1 and 2 are important, problem 3 is a major stumbling block for 22q13 deletion syndrome that I would like to address.
Clinical trials for 22q13 deletion syndrome are intended to treat defects or loss of SHANK3 (Kolevzon et al., 2014). The problem with finding a treatment for SHANK3 is just as Rachel – and many others – have described. If the subjects you are testing are too diverse, you will never see a clear impact of the drug you are testing. The subjects recruited for these studies have either SHANK3 mutations or have 22q13 deletion syndrome with terminal deletions of different sizes. This group is more diverse than many, perhaps all, of the other autism-related clinical studies that have failed. Going on past experience in the field, this clinical group will not provide useable results. Here are the reasons why.
SHANK3 mutations are complicated
Early on, there was hopeful enthusiasm about hunting for a cure for people with 22q13 deletion syndrome. At that time, SHANK3 mutations were lumped together with chromosomal deletions. Importantly, SHANK3 mutations were thought of as simply a loss of SHANK3 function. As it turns out, SHANK3 mutations are tremendously complicated. Different SHANK3 mutations can have very different effects on the gene, on the proteins it produces, on the neural development of the brain, and on the impact it has on both people and experimental animals. The most recent and most thorough review of Shank proteins (Monteiro and Feng, 2017) says it clearly: “Indeed, the idea that isoform-specific disruptions [different mutations] will result in different phenotypic consequences (and even result in different disorders) has recently gained momentum.” I can say with some pride that the momentum includes my June 2016 blog How to fix SHANK3, which makes that very same point. You cannot lump together people with different SHANK3 mutations and expect to get a single clear result.
Too few patients have the same SHANK3 mutation
To date, no one has been able to find enough people with the same SHANK3 mutation to do a drug study. You can find SHANK3 mutations in large autism databases, but these are not like a registry where you can call the patient up and ask them to participate. There is no doubt that medical researchers would pull together a SHANK3 drug study population, if they could. Autism is thought to be a polygenic disorder (like schizophrenia). Thus, we expect that many individuals from autism databases will also have mutations of multiple autism-related genes, not just SHANK3. Finding a large enough group of people with one (or two) SHANK3 mutations to study drugs will probably never happen.
Individuals with 22q13 deletions are too diverse
Another approach might be to use 22q13 deletion syndrome patients with terminal deletions that remove SHANK3 altogether. Every one of these patients would have exactly the same SHANK3 loss. Further, there is a registry for 22q13 deletion syndrome patients that might help with recruitment (PMSIR). While this seems appealing, it has its own flaw. Just as the SHANK3 mutation population is likely to have other autism and intellectual disability genes complicating the picture, chromosome 22 is full of genes that likely contribute to autism, intellectual disability, hypotonia and other phenotypic traits associated with SHANK3. Anyone who has read my other blogs has seen numerous examples of those genes (see Mouse models and How do we know which genes are important?). Because of the densely packed genes near SHANK3 (see Understanding deletion size), it is unlikely that a big enough group of people with 22q13 deletion syndrome can be found with deletions that don’t involve other critical genes on 22q13.
In her article, Rachel Zamzow discusses the N-of-1 Trials approach. We parents do this all the time. We experiment with different medicines on our one child. N-of-1 design simply has the clinical researcher follow the child during the test. I’m not a big fan of N-of-1. I prefer a mixed experimental approach where research animal testing is done in tandem with human testing (see Have you ever met a child like mine?).
In their detailed review of Shank proteins and autism, Monteiro and Feng recommended that “..careful genotype-phenotype patient stratification is required before individual testing of specific pharmacological agents.” That is, don’t test drugs until you understand the impact of the genes that have been lost. If you have been reading my blogs, that should sound very familiar.
Two things must change before we can expect drug testing to bring meaningful results. First, we need to organize Phelan McDermid syndrome, SHANK3 mutation syndrome(s), and chromosome 22q13 deletion syndrome into a meaningful “genotype-phenotype patient stratification”. That is, we need to define different types and subtypes of the syndrome that was once called 22q13 deletion syndrome. I proposed running an interactive session with parents and researchers in 2012, and for the session I put together a Power Point presentation called: “Defining PMS across Genotypes Phenotypes and Molecular Pathology.” I was asked not to present my ideas. Perhaps I will be given a chance, someday.
Second, we must spend the time to characterize the genes that are near SHANK3 on chromosome 22 and understand (in experimental animals) how they might contribute to 22q13 deletion syndrome. We need to study people with interstitial deletions, so we can isolate the effects of these genes. Efforts to explore the contributions of 22q13 genes has been lacking, yet they are a major impediment to the search for effective drug treatments.
22q13 deletion syndrome has left David completely dependent upon others for his day-to-day living. Both David and I have come to accept that. What we cannot do for David is know where it hurts when he is sick or injured. If I had one wish for a new medicine, that medication would let David point to where it hurts. That medicine, or any useful medication, is not going to happen until someone takes the needed steps to remove the impediments that interfere with productive drug testing. It is clear where we need to go. The question becomes, who will take us there?
One nice thing about writing a blog is getting feedback. While I embrace and benefit from all kinds of feedback, I admit being partial to positive feedback. I get some nice comments from friend on my Facebook page. I love the encouraging comments that get posted here. There is another kind of feedback that is neither positive nor negative, but very informative. It is the kind of feedback that we should all pay attention to. Visitors vote with their mouse (or touchscreen). Every time someone clicks on a blog link, WordPress adds one to a blog page counter. Now that it is 2017, let’s see what the numbers recorded in 2016 tell us.
The most requested page is at the bottom of the graph: How do I know which genes are missing? That is the number one question on parents’ minds. It makes sense. If your car breaks down, you want to know what caused it, even if you don’t know much about cars. At the very least, you have some idea of what needs fixing.
Three more questions are virtually tied for 2nd place. Have you ever met a child like mine? and How to fix SHANK3 are discussions of SHANK3 and its relationship to the other genes of 22q13 deletion syndrome. Together, with the most requested blog page, over one-third (35%) of mouse clicks on this blog are from people who want to understand how all the genes of 22q13 deletion syndrome operate together to produce the disorder. The other blog page that is tied for 2nd place addresses the same topic from the opposite direction: How can the same deletion have such different consequences?
Taken together, nearly half (46%) of the information people want from this blog is to understand what genes are missing and why those genes matter.
Most visitors in 2016 already knew about 22q13 deletion syndrome. Only about 4% of all clicks went to 22q13 deletion syndrome – an introduction. Fewer clicks went to learning about the author. (I can live with that!) But, I think there are a other links that deserve attention. Here is a suggestion for the new year:
Gene deletion versus mutation: sometimes missing a gene is better. SHANK3 is not the only gene of 22q13 that can have serious consequences when mutated (modified, but not lost altogether). Much is said about SHANK3 mutations, but 98% of people with 22q13 deletion syndrome are missing SHANK3 altogether. Understanding the difference may be crucial to finding cures.
I have dedicated the past few months to formal writing about 22q13 genes aimed at the scientific community. That work has taken me away from this blog, but, hopefully, taken us all closer to effective treatments for our children. That work is done for the moment and I hope to get back to this blog on a more regular basis.
Anyone who has read most or all of my blog pages knows that my goal is to help parents, scientists and other members of the 22q13 deletion syndrome community understand how the genetic landscape of chromosome 22 must shape our thinking if we are going to realistically pursue treatments. If you have not read the earlier blogs, much of this one may seem foreign. This blog is based heavily on prior ones. Because of the overlap, I will omit scientific references and simply recommend reviewing prior posts for supporting evidence.
There has been a recent flurry of mouse model papers on the Shank3 gene. The number of model mice has passed one dozen. People who work on Shank3 mice love to describe their rodents’ behaviors as mouse analogs to human behaviors. When an unusual mouse behavior is “rescued” with a chemical compound, the implicit (sometimes explicit) suggestion is that mouse research is on a path to curing autism, “Phelan-McDermid syndrome” (PMS) and maybe even schizophrenia. Some researchers like to define PMS as a disturbance of SHANK3, which guarantees that any SHANK3 fix will fix PMS, whether or not the child is any better. I am not going to argue with this rosy, perhaps fanciful, view of current rodent research. It helps patients’ families feel hopeful and keeps funding and publications flowing. These are good things that a more conservative interpretation of the data might never accomplish.
From a practical standpoint, however, we still need a strategy for fixing SHANK3 problems in humans. We need a plan that has more to do with the human disorder than the rodent one, and more to do with therapeutic benefit than a detectable statistical change. The plan needs to be based on what we know more than what we speculate. The plan needs to be about the patients, not the scientists, funding agencies or feel-good charity organizations.
The first thing we don’t know is whether human SHANK3 mutation causes the same problems as SHANK3 deletion. Numerous rodent studies speculate that the influence of Shank3 mutation is a “dosage effect”. That is, the effect is simply due to how much SHANK3 protein is lost. Yet, total removal of all SHANK3 protein from a mouse has less effect than many Shank3 gene mutations. Among the many different Shank3 mutations studied in mice, the behavioral, molecular, electrophysiological and drug effects differ widely. This “diversity of phenotypes” is the hallmark of a mutation syndrome, not simply a dosage effect. In other words, in rodents there is a Shank3 mutation syndrome that is different from Shank3 deletion.
What about humans? Is SHANK3 mutation different from SHANK3 deletion? Well, no one knows, because only one patient has ever been described in the published literature as having a complete SHANK3 deletion without also damaging or removing other well-established brain genes, and the published information on that patient is limited. It should not be necessary to emphasize this, but it make no sense to talk about exquisitely, selectively removing exactly one gene in a mouse and comparing that to humans missing 20, 30 or 100 genes. Any study based on a mouse model that accidently knocked out 2 or 3 nearby genes would never get published. It is disingenuous to insist on precision mouse gene editing and then make comparisons to patient populations that are nearly devoid of matching examples. It is inconvenient that we don’t have clean human examples, but we parents of 22q13 deletion syndrome children deal with a lot of inconveniences that we cannot wish away. In that regard, we are not very sympathetic to wishful scientists.
So, let’s be clear on what we don’t know. We don’t know if selective SHANK3 deletions are different from SHANK3 mutations in humans. However, we do know that humans with different SHANK3 mutations can have very different presentations, including autism spectrum disorder (ASD), intellectual disability (ID), combined ASD with ID, and combined schizophrenia with ID. So, we know that the diversity of phenotypes associated with mouse Shank3 mutations parallels the diversity of human phenotypes. This parallel gives us some, albeit weak, evidence that the effects of human SHANK3 mutations are not a simple a dosage effect. We are still limited by the paucity of human cases to assess the real impact of a pure SHANK3 deletion.
Wishful thinking aside, let’s go with what the (limited) evidence says: human SHANK3 mutations (including deletions that disrupt the gene) probably have effects other than reducing the availability of SHANK3 protein. Because mutation syndromes are not uncommon on chromosome 22 and elsewhere, there is ample precedence for understanding how mutations can disrupt normal function. The mouse (and human) Shank3 gene has 7 intragenic promoter regions and an estimated 20 to 100 natural isoforms (variants of the protein produced). The SHANK3 protein is very similar to SHANK1 and SHANK2, with many molecular binding partners in common. That is, the three shank proteins all interact with essentially the same molecules in the neurons of the brain. Taking a reasonable speculative leap, mutation of SHANK3 gene can produce some or many SHANK3 fragments that wreck havoc with the assembly of the synapse. As an analogy, think about placing a bunch of defective nuts and bolts into the manufacturing process for a car or airplane. The production line is better off substituting different hardware (e.g., using SHANK1 or SHANK2) than installing parts with defective hardware (broken bits of SHANK3). The somewhat unexpected conclusion is that we might be able to treat disorders of SHANK3 mutation by shutting down the SHANK3 genes partially, or altogether. This approach can be tested in mice.
If we are considering SHANK3 deletion as a treatment for SHANK3 mutation, then we better be prepared to treat SHANK3 deletion. I believe recent results from the first Shank3 complete knockout mouse provides a path for understanding and treating human SHANK3 deletion. The most abiding and measureable effect of complete Shank3 deletion in the mouse is failure to engage and benefit from an operant conditioning task (lever pressing for a reward). This effect appears to be associated with abnormal ventral striatal function, which is consistent with many previous studies of the ventral striatum. Failure to explore and learn would be indicative of ID in humans, so it is of great interest to understand the exact relationship between the learning deficits in humans with pure SHANK3 deletions and mice with pure (complete) Shank3 deletions. Such an undertaking would require a very modern and somewhat novel strategy in the world of pre-clinical neuropsychiatric research.
The precise nature of the mouse learning deficit is not yet understood. Learning is a complex process and many aspects are very subtle. Even the reported rescue of learning in the Shank3 knockout mouse creates more questions than answers. These questions go to the heart of how SHANK3 loss might contribute to intellectual disability in humans. How can the details of learning deficits caused by SHANK3 deletion be dissected out? Given the rarity of pure SHANK3 deletion, I propose that a single subject (or two) be invited as true participants in a scientific study of their learning abilities, and that the latest computational approaches (often used in animal research) be applied in a series of iterative testing to model and measure the learning deficits.
Current, state-of-the-art scientific learning studies are computationally based. Learning tasks are designed to incorporate variables that can be directly tied to equations describing an underlying theoretical framework of the learning process. Animal researchers are adept at designing learning tasks in ways that do not require verbal instruction. They are equally practiced at inferring the results without the need for verbal reports. Still, with the participation of a fluent verbal subject, researchers can work with the subject to help design tasks (games) that are interesting and engaging. Rewards for mice are often in the form of sweetened concentrated milk droplets. For healthy adults, money is commonly used as an incentive. The SHANK3 deletion participant may prefer to see dancing fairies or a music video clip.
As these learning tasks begin to characterize the nature of the deficit seen in the subject/participant, they are then re-designed for testing in animal models. Current rodent models can be used, but there is no reason the same tasks cannot be explored in nonhuman primates for fMRI and electrophysiological investigation. The technology of gene editing, common in mice, has reached farm animals and at least two species of nonhuman primates. As these methods become more mainstream, complete SHANK3 deletion could be a practical research option, especially in old world monkeys, species that shares important common features with human cortical evolution.
The goal of this scientist/participant research partnership is to develop a sensitive cross-species measure of learning ability that parametrizes the impact of SHANK3 dosage. Such a measure provides two invaluable assets to the development of treatments. First, animal models can be validated (or not) based on exquisite computational approaches that may be able to distinguish species differences from the influence of SHANK3 dosage. Second, interventions, either learning-based or pharmaceutical, can be tested using measures sufficiently sensitive to reflect the identified nature of the deficit. What can this human research/animal research partnership hope to produce? The first successes may be refinements to educational methodologies. The learning models could point the way to improvements in teaching strategies. Later, dare we hope, may be pharmaceutical interventions.
Wouldn’t it be splendid if parents could continue to hope, scientists could continue to get published, feel-good organizations could continue to raise money, and in the meanwhile, our kids could get better, too?
Jannine Cody, the parent/scientist who studies 18q deletions, says that since every deletion is different, every child with a deletion is different. At the PMS family conferences we met other children with 22q13 deletion syndrome who, at the time, had striking similarities with David. These children had chromosome 22 deletions of various sizes, and similar children did not always seem to have the same size deletions. We know now that genes are not distributed equally along 22q13, so children with small deletions can be quite different from each other, and children with large deletions can be quite similar (see Understanding deletion size). We also know there are good scientific reasons to expect differences (see How can the same deletion have such different consequences?). Some things are pretty obvious after a while. The kids who could not walk or talk generally had larger deletions. Those with larger deletions also had many more medical problems. Obviously, more genes lost means more problems. Regular readers of this blog have seen evidence of why it is very important to know which genes are missing (see How do I know which genes are missing?).
Some people feel that research on 22q13 genes should be done one gene at a time, starting with SHANK3. I am not a big proponent of this approach, since it ignores a lot of research already done on ARSA, MAPK8IP2, CHKB, CPT1B, PANX2, ALG12, BRD1, SULT4A1 and other genes known to cause disorders in humans, mice or both. The one gene-at-a-time approach also slows research by making one gene sound much more important than others. It seems to me if we spend 5 to 10 years on each gene, we are doomed to spending 500 to 1,000 years. If that sounds pretty absurd, well, it is. Maybe it will only take 200 years to do it this way. That still seems too long to me. That is why I recommend the scientific program be managed by someone with a deep understanding of science leadership (see 22q13 deletion syndrome and science leadership). The “SHANK3 or bust” research program has succeed in some ways. Recently, after about a dozen mouse models of Shank3, there is a new mouse with the first complete deletion of the gene. All the other mice were various examples of gene mutation. As we know, the effects of mutation (or removing part of the gene) can be very different from deletion (see Gene deletion versus mutation: sometimes missing a gene is better). This is critically important! The main reason for supporting Shank3 mouse research is the argument that most (not all) patients are missing the SHANK3 gene entirely. Thus, it is SHANK3 deletions that make the research important to our families. (Note that mouse Shank3 mutation research has a very separate goal: understanding how mutations might contribute to general forms of autism.)
So, we now have a real Shank3 deletion mouse and everyone is very excited about it (Mouse Model of Autism Offers Insights to Human Patients, Potential Drug Targets). Of course, be skeptical of what the university PR team says (see Mouse models). Let’s take a look at this first-ever complete Shank3 knockout mouse. First off, the major finding is that this mouse is different from the many mutation mouse models. No one should be surprised. What is surprising is that you have to completely wipe out 100% of Shank3 to see a measurable difference between these mice and normal mice. Even more shocking is that these mice are walking around, playing with other mice, eating, talking mice talk (ultrasonic sounds) with no shank3 whatsoever in their bodies! The mice missing 100% of Shank3 are different from other mice, but mice missing 50% are not different in any measurable way. Note that humans with 22q13 deletion syndrome are missing only one of the two genes and best evidence is that they have lost only about 25% of their shank3 protein (See this research paper).
So, is there something wrong with the mouse study? Are mice just way different from humans, or is there another explanation? Maybe it all makes sense. Have you ever met a human missing all of SHANK3 and only SHANK3? The complete knockout Shank3 mouse is best compared with a person like that, someone who is not missing any other genes and has no known mutations. It is not good enough to have someone with a “small deletion”, since there is strong evidence that adjacent genes impact brain function. This mouse models SHANK3 deletion. I have met only one person who seems to fit this description.
Phelan McDermid syndrome is characterized by developmental delays, moderate to severe intellectual disability, little or no expressive language, and infant hypotonia (floppy baby syndrome). Some people argue that the syndrome is also characterized by a high incidence of autism spectrum disorder, although some top scientists disagree. The person I met was probably never a floppy baby, has practically normal speech, and that person has no evidence of autism. Rather, the person I met has some problems with coordination, has a great difficulty learning and is socially a wonderful person to meet and engage with, perhaps to a fault. Tragically, like all of our children, that person will never navigate the world well enough to live an independent life.
In summary, when I read the scientific paper on the complete Shank3 knockout mouse, what struck me was how many tests the complete, 100% knockout mouse passed without demonstrable evidence of a problem. Mice missing one copy are normal in almost every test. Mice missing both copies are not “normal”, but clearly, even these mice are nothing like my son.
How important is SHANK3? It is impossible to make that judgement based on only one clinical case. The person I met has lost all independence for that person’s entire life. That is very important. Moreover, it is tragic. But for 95% of families, 22q13 deletion syndrome comes with the full set of core features of 22q13 deletion syndrome. David cannot tell me when he feels sick, where it hurts, or if he was mistreated in his group home. It took him 6 years to overcome his floppy baby syndrome enough to walk and three more years before he could eat by mouth. His autism-like features interfere with social contact.
As of now, the most parsimonious explanation of what we know is that SHANK3, alone, does not produce the core features of 22q13 deletion syndrome. It is a contributor in most, but not all, cases.
There have been a number of press releases and feel-good articles circulating among my 22q13 deletion syndrome Facebook friends celebrating the advancement of mouse models of “Phelan McDermid syndrome”. I am all for enthusiasm! However, cheering is more fun if you know how the game is played and what to cheer for. With that in mind, I would like to look carefully at models. What is a good model and what might it tell us?
I started out in engineering, where modeling is very important. Let’s imagine modeling an airplane. The first issue of modeling is, what aspects of the plane are motivating us to build a model? In this case we might want to learn about how the airplane will perform if the tail is damaged in flight. We can construct a scaled-down miniature with wings, tail, etc. and omit the inside furnishing (cockpit, storage compartments, etc.). If the miniature has the exact same shape and moving wing/tail parts as the real airplane, we would say our model has construct validity. That is, it is constructed in a way that reflects the original plane. The next step is to put our model into a wind tunnel and see if it flies. The model is held on a wire. We can adjust the wing flaps and other control surfaces. If the model tries to rise at the same wind speed as the real plane, and tries to bank left or right with the same amount of wing flap adjustment as the real plane, we can say this model has face validity. That is, with regard to what we are testing, the model behaves much like the real plane. We are finally ready to benefit from all our work. Let’s take the model for a test flight and then poke a hole in the tail. What settings of the flaps and other control surfaces allow us to keep control of the plane? We may struggle with this for weeks or months hoping we can learn to control our crippled model plane. If we find a solution, maybe we have found a way for pilots to rescue their plane in the event of a similar emergency. If this scheme works, our model has predictive validity. Thus, we measure a model’s worth by:
does it model what we want?
is it constructed in a way that tests what is important to us? (construct validity)
does it perform in a way that mimics what we already know? (face validity)
will model manipulations tell us how the real thing will respond? (predictive validity)
I hope you are getting a picture of what an animal model should do. Let’s look at a mouse model of “Phelan McDermid syndrome” or “PMS”. I use quotes because different scientists have different definitions of “PMS”. See Introduction. For this blog, I will omit the quotes, but remember that there are numerous definitions floating around.
The definition of PMS is important for modeling. The definition of PMS tells us what people claim to be modeling. Some scientists define PMS as a deletion or mutation on chromosome 22 that involves SHANK3. That is fine with me, but that omits the rest of 22q13 deletion syndrome, since there are numerous cases of “interstitial deletions” that don’t affect SHANK3. So, SHANK3 might not be a good choice of model for many families. It depends on the deletion and it depends on what is causing your child the greatest difficulties. There are currently mouse models for 11 different PMS genes for deletions of 1 Mbase or larger. Every gene on this list is relevant to 95% of patients (See Understanding deletion size.) The 11 genes are BRD1, CHKB, CPT1B, MAPK8IP2, MAPK11, MAPK12, NCAPH2, PANX2, PIM3, SHANK3, TYMP. Most of these mouse models are very well studied. If you go further up the chromosome you find other well studied genes with mouse models, like ATXN10. So, the choice of gene is all about what aspect of a disease or syndrome you wish to study.
SHANK3 is popular not because of PMS. It is popular because is has been associated with autism. There are under 1,000 identified people in the USA with PMS, but there are an estimated 36,500 children born each year in the USA with autism. Parents of 22q13 deletion syndrome children should appreciate that researchers who study Shank3 mice are tapping into the national (and world) autism crisis. Our children are a convenient source of subjects, which is why the big national study officially excludes PMS families with interstitial deletions that do not affect SHANK3. Children with SHANK3 mutations are of greatest interest even though they technically do not have 22q13 deletion syndrome (that is why the name PMS was created). Note that only 1/3 of children with PMS have autism, so patients with SHANK3 mutation and autism are the most valuable research subjects.
Although SHANK3 is of great current interest, autism is caused by hundreds of genes. Most parents don’t realize that many 22q13 genes are autism-related or suspected to contribute to autism. Some of the autism genes on chromosome 22 are BRD1, CELSR1, CHKB, MAPK12, PANX2, BRD1. Further up the chromosome (associated with somewhat larger deletions) you can find CELSR1, WNT7B, TCF20, EP300 and others.
Now we understand that Shank3 mice need to be models of something. From the above lists of genes, it is pretty clear that a mouse missing only one gene is not a universal models of either 22q13 deletion syndrome or autism. Both conditions involve a large number of genes. The Shank3 mice are single gene models. The mice are fashioned after super-rare cases of people with specific SHANK3 mutations. Let’s see how these models stack up in terms of construct, face and predictive validities.
All of the published papers so far describe Shank3 mutations and microdeletions, not deletion. I am not going to cite the specific papers here. I have written brief reviews on most of these papers. Contact me if you would like specifics. Some of the mouse Shank3 models use deletions that reflect mutations found in actual patients. For those specific patients (often just one or two), the genetic manipulations have construct validity. That is, the mouse gene has been changed in a way very similar to the human gene. As for the rest of us PMS parents, 95% of our children are missing the SHANK3 gene altogether, along with 30 to 100 more genes. So, these mice do not have very strong construct validity for our children. It is well known that mutations can be very different from deletions. (see Gene deletion versus mutation.) Of course, mutating a single gene may not be very helpful to understanding your child when so many other important genes may be involved. (see How do we know which genes are important?) There is also the problem of using a mouse to model a human. The gene is mutated in an animal that lacks brain areas that are crucial to human behavior, like the granular prefrontal cortex. If these brain areas are important to autism, then the construct validity is weaker.
To be fair, a lot of work goes into to creating a knockout mouse. I don’t have first-hand experience, but I work down the hall from a colleague who is an expert. He works hard and I can read the frustration in his brow on tough days. Just making a mouse is not good enough, you have to prove you have modified the right gene in the right place without messing up the rest of the genome. Then you cross-breed, back breed and then do more validations. My hat is off to those people who make a living this way. When done, the mouse has construct validity in that the targeted gene has been modified.
Face validity is a huge problem with mouse models. Our model airplane ascends and descends, banks and aerodynamically behaves much like its real-life big brother. We know the flight behaviors that are important and we can directly (although not perfectly) compare our model plane to the real thing. How do we compare our model disease to the real disease? Generally, the first step is to compare normal mice to our genetically modified mouse. We note what is different between the two. Then, we compare normal human subjects (“developmentally typical”) to those with the syndrome or disorder. The question then becomes, do the mouse differences seem to reflect the human differences? Mouse models of kidney function and cancer have been very successful with face validity. Urine output and tumor size are easier to measure than social behavior and eye contact in mice. Biopsies of human kidney and many tumors are also much easier than brain biopsies. It is no surprise, then, that mouse models of neuropsychiatric disorders are hard to validate.
The differences between normal and mutated mice can be observed in brain structure, chemical signatures, cellular changes and gene expression. For the most part, there is very little human data for comparison. There are studies with human post mortem tissue that can be helpful, but most of that tissue is from normal human brain. As you might guess, screening for gene expression and other changes in a donated brain from a PMS patient will have issues. Most patients have too many genes involved. One helpful approach is induced pluripotential stem cells (iPScs), grown from skin samples or hair follicles. One might even be fortunate enough to find a donor who’s genetic mutation matches the mouse (or vise versa). However, iPScs do not undergo normal development, so developmental studies are impossible at the moment. They also don’t produce circuits like an intact brain. These are all limitations that impact face validity.
The one common feature you can examine between mice and humans is behavior. There are two extreme viewpoints in this regard. At one extreme, some scientists argue that any behavioral difference between the normal mouse and the genetically modified mouse is “autism-associated” because of the construct validity. At the other extreme, a few scientists argue that the mouse behavioral difference should reflect the standard manual that defines neuropsychiatric disease (DSM-5). The remaining scientists are in the middle until it comes to publishing a paper. Then, they make the argument that their animals’ behavior somehow reflects the behavior of a person with autism. It is my observation that the more popular or prestigious the journal, the more their mouse sounds like a miniature human.
A major problem with Shank3 mice is that, almost universally, they don’t have a significant aberrant behavior. How can you study behavior when it seems normal? There are two solutions, but both have problems. With one approach, researchers find a mutation spot on the gene that is extra good at producing a pathological behavior (e.g., ignoring other mice, not learning, rubbing its fur until the skin is damaged). Another approach is to mutate both copies of the gene. Each of these solutions creates a new problem. In the first case, we are no longer modeling PMS or autism, because this behavior only shows up in very specific mutations. In the second case, we have lost construct validity. We were never trying to model mutation or loss of both copies of SHANK3. That would likely be a different syndrome (just as mutation is different from deletion).
In the end, current models of SHANK3 mutation (autism and SHANK3 mutation aspect of PMS) provide information about molecules and neurons, but are very limited models of the human diseases. They don’t necessarily say much about 22q13 deletion syndrome in general. It is interesting to note that mouse models of other genes of 22q13 deletion syndrome may be more promising. Several mouse models have clear consequences with only one gene mutated or deleted. For example, the Cpt1b mouse model is used to study cold tolerance and insulin sensitivity, and the autism and schizophrenia-associated gene, BRD1, is under intense research right now by a group in Denmark. In both cases the behavioral effects are clear when only one copy of the gene is affected.
We are not done yet, because the most important and most difficult type of validity is predictive validity. That is, does your mouse model tell you what will happen in your human subjects? Given all the problems with making a mouse model of a neuropsychiatric disorder, it is no surprise that the models have not lead to translational (treatment) gains. (See Hope for autism treatment dims as more drug trials fail).
I have heard many people in the research field try to sound hopeful to parents. I believe that hope, when anchored in reality, is very helpful and important. But, too many people have alternative reasons for fanning the flames of hope by knowingly or unknowingly misrepresenting the size of strides in research. My recent blog (How can the same deletion have such different consequences?) touches on why so many parents are mislead by researchers, writers and support organizations. The subsequent blog (22q13 deletion syndrome and science leadership) explains that it does not have to be this way if we bite the bullet and hire qualified leaders.
As a parent, my heartstrings feel the tug of hope every day. The urge to follow those feelings is managed by having a deeper understanding of the research world, and remembering that too many people are eager to give those strings an extra tug.