Splitting, Lumping and Clustering

David circa 2009
David happily watching his videos in the summer of 2009

If we want to find treatments for Phelan McDermid syndrome (PMS), first we need to figure out what is PMS.  That was spelled out in my blog: Why don’t we have better drugs for 22q13 deletion syndrome? My next blog addressed how to organize all the different genetic deletions and mutations so that we can define PMS (Defining Phelan McDermid syndrome).  Today’s blog addresses ways we can define different types of PMS.  If we don’t define different types, we are wasting our time experimenting with treatments.  For instance, some PMS kids talk fluently, some talk in short sentences, some can only say single words and many, like David, do not talk at all.  These and many other difference warrant different groups of kids when we test treatments.

Just as there is huge variation in abilities and behavioral characteristics, our kids have very diverse genetics.  Recent studies of rodents  show that not all with Shank3 mutations are alike.  In fact, drugs may work very differently on different Shank3 mutations. Anyone who has kept up with my blogs knows that deletions of different genes are likely to have very different effects on our children.  These difference are very important.

Useful drug testing is stuck right now until we develop a way to categorize people with PMS based on both phenotypic characteristics (symptoms and manifestations) and genotypes (deletions versus mutations and which genes are affected).

I have heard scientists who study Shank3 mice talk about “splitting” and “lumping”.  Splitting is breaking groups into subgroups.  Lumping is putting everyone/everything together into a single group.  Lumping has not worked and the growing consensus is that lumping will never work in our population. Splitting based on just one characteristic (e.g., deletion size) probably won’t work, either.  We need a more refined approach.  What we need is “clustering”.  Clustering is what mathematicians and scientists do when categorizing requires using many different characteristics at once.

Here is an example.  Let’s say you want to buy a car.  You might look at various cars and think about both price and gas milage.  You could make a graph something like this:

Car Clustering2Similar types of cars have similar prices versus gas milage tradeoffs.  Race cars are more expensive, but get poor gas milage. Clustering is when you identify meaningful subgroups on a graph because the individual points are close together.  Each group is a cluster.  Even if not every car fits neatly into a cluster, you still have an organizational scheme that can be very helpful.

PMS needs meaningful groups.  Clustering can get complicated when there are more and more features that divide up the population.  However, computer programs can take care of the complexities.  What we need first is to identify which characteristics are important for grouping.  As a practical matter, researchers go back and forth. They consider characteristics, run a program that automatically clusters the data based on those characteristics, and then look to see if the clusters make sense.  That is what we need to do.

When we took David to the PMS Foundation Family Conferences in 2008 and 2010, we met a handful of kids that were remarkably like David (see photo of David, above).  What was it about those kids?  As I recall, they walked the same way, loved watching music videos, asked for help the same way, were nonverbal and all have relatively larger deletions.  Are those meaningful characteristics?  Will they help us divide PMS into different groups for meaningful drug studies? We need to find out.

arm22q13

 

Previous blogs

Defining Phelan McDermid syndrome
Why don’t we have better drugs for 22q13 deletion syndrome?
What do parents want to know?
Is 22q13 deletion syndrome a mitochondrial disorder?
Educating children with 22q13 deletion syndrome
How to fix SHANK3
Have you ever met a child like mine?
How do I know which genes are missing?

Mouse models
Science Leadership
How can the same deletion have such different consequences?
22q13 and the hope of precision medicine
22q13 Deletion Syndrome: hypotonia
Understanding gene size
Gene deletions versus mutations: sometimes missing a gene is better.
Is 22q13 deletion syndrome a ciliopathy?
Understanding translocations in 22q13 deletion syndrome: genetics and evolution
Understanding deletion size
Can 22q13 deletion syndrome cause ulcerative colitis?
Can 22q13 deletion syndrome cause cancer?
22q13 deletion syndrome – an introduction

Defining Phelan McDermid syndrome

david-and-dad-4-march-2017-small
It is all a matter of how you look at the problem.

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.

definition-of-22q13

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:

relationship-between-shank3-and-pms

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 SHANK3 mutations 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.

 

arm22q13

Previous blogs

Why don’t we have better drugs for 22q13 deletion syndrome?
What do parents want to know?
Is 22q13 deletion syndrome a mitochondrial disorder?
Educating children with 22q13 deletion syndrome
How to fix SHANK3
Have you ever met a child like mine?
How do I know which genes are missing?

Mouse models
Science Leadership
How can the same deletion have such different consequences?
22q13 and the hope of precision medicine
22q13 Deletion Syndrome: hypotonia
Understanding gene size
Gene deletions versus mutations: sometimes missing a gene is better.
Is 22q13 deletion syndrome a ciliopathy?
Understanding translocations in 22q13 deletion syndrome: genetics and evolution
Understanding deletion size
Can 22q13 deletion syndrome cause ulcerative colitis?
Can 22q13 deletion syndrome cause cancer?
22q13 deletion syndrome – an introduction

How to fix SHANK3

david-eating-cerial-bars
David snacking on some cereal bar bits
Aubree and Mickey rev 2
Human – Mouse Partnership

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?

 

arm22q13

Previous blogs

Have you ever met a child like mine?
How do I know which genes are missing?

Mouse models
Science Leadership
How can the same deletion have such different consequences?
22q13 and the hope of precision medicine
22q13 Deletion Syndrome: hypotonia
Understanding gene size
Gene deletions versus mutations: sometimes missing a gene is better.
Is 22q13 deletion syndrome a ciliopathy?
Understanding translocations in 22q13 deletion syndrome: genetics and evolution
Understanding deletion size
Can 22q13 deletion syndrome cause ulcerative colitis?
Can 22q13 deletion syndrome cause cancer?
22q13 deletion syndrome – an introduction