Summary of "Shank3 and Phelan-McDermid Syndrome" Mini-symposium

By Andy Mitz and Paulina Rychenkova

Philadelphia, PA

May 19, 2010

The meeting took place on May 19, 2010, 1-5 pm at the Center for Autism Research at the Children's Hospital of Philadelphia (CHOP).

It was organized by Dr. Joseph Buxbaum (Director, Seaver Autism Center, Mount Sinai School of Medicine), who invited the presenters and coordinated schedule; by Dr. Robert Schultz (Director, Center for Autism Research, CHOP) who provided meeting facilities, and by the Phelan-McDermid Syndrome Foundation who encouraged participation from affected families and sponsored the post-meeting dinner for families, speakers and VIPs.

The meeting drew 45 participants including:

21 researchers, including 6 speakers;

15 family members representing 10 families;

2 members of the Interim Scientific Advisory Committee;

2 representatives from Pfizer;

1 representative from Autism Speaks; and

4 others.

Below are brief summaries of the six presentations:

Katy Phelan, PhD (Molecular Pathology Laboratory Network Inc.) "Cytogenetic and clinical characteristics of Phelan-McDermid Syndrome"

Dr. Phelan described in detail the Phelan-McDermid Syndrome (PMS) phenotype and the range of observed symptoms. She described observed genetic findings and the types of studies to diagnose PMS used in the past and present.  She showed that so far no correlation has been observed between the genotype and the phenotype of an individual with PMS, but a study involving up to 100 individuals was underway at Greenwood which will attempt to find such correlation. She mentioned that PMS is in great majority of cases a result of a mutation in the Shank3 gene (such a mutation may be a deletion of one copy of Shank3, a deletion that disrupts Shank3, or a mutation in the Shank3 gene itself).

Dr. Phelan listed the deletion types and their frequency.  In order of frequency: simple, translocations, interstitial deletions, gene frame shift, duplications. She also requested and received very helpful comments from Dr. Curtis Rogers, who has the world's greatest medical experience with PMS children and adults.

Catalina Betancur, PhD (INSERM, Paris, France) "Spectrum of SHANK3 mutations in Phelan-McDermid syndrome and autism"

Dr. Betancur described a range of syndromes where a subset of patients received autism spectrum diagnoses (ASD), with PMS being one of the syndromes. She described a study of over 650 families with autism who sought to identify underlying genetic cause. She described a unique case of a family where both parents (reportedly healthy) presented with duplication of Shank3, with two children. Both children also had duplication of Shank3 and had severe autism and mental retardation.  Studying this highly unusual case further may lead to new understanding of the role of Shank3 and wide range of phenotypes observed in PMS.

Dr. Betancur showed a slide with 30 to 40 medical conditions known to be associated with autism, which accounts for 10-25% of known cases of autistic spectrum disorders. Most of these conditions are genetic. All genetic causes produce intellectual disabilities (formally known as "mental retardation" ).  Dr. Betancur provided an excellent review of the evidence pointing to SHANK3 as the causal agent in PMS.  Using a "critical region analysis", she showed that only one other gene (RABL2B) may be contributing to PMS in those patients with the smallest deletions.  The Paris Autism Research International Study (PARIS) studied 663 families and screened 97 for deletions and 227 for SHANK3 mutations.  Three cases of SHANK3 mutations were discovered.  The duplication cases lacked penetrance (that is, some people show symptoms, some do not).  The people with duplication included an individual with Aspergers who had precocious language.

Dr. Betancur also discussed the first two cases ever of SHANK2 deletions. In one case language was delayed, in the other case language was seriously delayed, but in both cases language was functional.

Carlo Sala, PhD (CNR IN, Milano, Italy) "The role of Shank proteins in brain function"

Dr. Sala gave an overview of the Shank family of proteins showing their structure.  He described the role played by Shank proteins in excitatory and inhibitory synapses, and showed in full complexity the web of interactions that Shank proteins are involved in highlighting their role as "scaffold" proteins.   He focused on Shank1, its interaction with Homer proteins, and its role in brain function.  He described the role of Shank3 protein in neurons, specifically that in neurons knocked down for Shank3, dendritic spines are reduced in number and dimension.  He showed that mGluR5 is reduced in neurons knocked down for Shank3. All of the above studies were done in mouse neurons. He mentioned but did not elaborate on three potential avenues for therapeutic intervention possible for PMS.  He mentioned that his lab is also creating iPS (induced pluripotent stem), cells from fibroblasts of PMS patients, as well as patients with another rare syndrome (not specified). He mentioned during break another project underway at his lab: using lentivirus to transfer the missing gene into brain of a knock-out mouse, this was being done for the other rare syndrome, not for PMS.  Apparently Shank3 is too large a protein to be transferred using lentivirus.

Dr. Sala's lecture was very technical and without a PA system in the room his talk was difficult to hear. However, as the most experienced SHANK researcher in the room, his lecture provided the most insight into research options.  Dr. Sala explained that SHANK scaffolding proteins are important because they "recruit" many other important synaptic proteins. This may explain some of Dr. Buxbaum's results (see below).  He compared SHANK1 to SHANK3.  An excess of SHANK1 can make existing spines more mature. In contrast, an excess of SHANK3 can create an excess of spines.  This is important in interpreting other people's work. Dr. Sala helped create the first SHANK1 mouse and found, much to everyone's surprise, that SHANK1 mice learn FASTER than normal (aka "wild type") mice. On the other hand, SHANK1 mice do not retain their newly learned skills.

Dr. Sala identified two important potential avenues for therapeutic intervention. SHANK3 is regulated through DNA methylation and SHANK3 is important for pERK1/2 activation. Drugs may be effective through either or both of these pathways.

During the question period Dr. Sala noted that, although SHANK3 is directly involved in regulating NMDA and mGlu receptors, it is not directly involved in the regulation of AMPA receptors.  (see below).

Joseph Buxbaum, PhD (Seaver Autism Center, Mount Sinai School of Medicine) "Haploinsufficiency of Shank3 leads to impairments in synaptic transmission and synaptic plasticity"

Dr. Buxbaum described findings from neuroanatomical studies done on a heterozygote (missing one copy) Shank3 mouse (het Shank3) which was created by his lab.  He focused on two measures: Long-term potentiation (LTP) and morphology of dendritic spines.  LTP is a measures of synaptic plasticity and is widely considered to be one of the major cellular mechanisms underlying learning and memory.  He showed that LTP was reduced in het Shank3 mice as compared with wild-type mice.  He studied morphology of spines in het Shank3 mice showing thinner spines and reduced number of connections (i.e. relatively immature spines) early in development as compared with wild-type animal. This difference appeared to go away over time, i.e. spines of het Shank3 mice caught up over time.  He concluded that AMPA receptor complex was impacted by the lack of Shank3 protein.  He used a derivative of insulin-like growth factor 1 (IGF-1) in a two-week treatment of het Shank3 mice.  He showed that deficits observed in LTP and in spine volume were corrected following the two-week treatment.  He also showed improvement following the same treatment course in knock-out Shank3 (missing both copies of Shank3) mice.  He observed that IGF-1 or like compounds may present a potential therapy for PMS patients, following further verification of results and clinical trials in human subjects.

Although Dr. Buxbaum was able to show partial "rescue" of LTP effects in both het Shank3 and complete KO mice, the drug that was tested was targeted directly at AMPA receptors. Dr. Sala's work (see above) showed that AMPA receptors are not directly regulated by Shank3, so there is some question whether this class of drugs will be useful for PMS.  The transient spine expansion measurements (spine size Vs time) plotted by Dr. Buxbaum showed impairments in both early and late phases of the spine expansion. This suggests problems with both receptor trafficking (early phase) and PSD stabilization (late phase).  Will two different drugs be necessary? Only time will tell. Dr. Buxbaum was very careful to remind the audience that many of his results are preliminary, and many drugs may need to be tested.  (We should also keep our fingers crossed!)

Jacqueline Crawley, PhD (NIMH) "Mouse behavioral assays relevant to the symptoms of autism"

Dr. Crawley reviewed types of studies that are done on mouse models of autism (so-called BTBR mouse) and types of results observed around social behavior and vocalizations.  She described results of several preliminary studies done on Shank3 mice.  These studies showed decreased male-female social interaction, decreased ultra-sonic vocalizations on presence of other animals, and unchanged olfactory habituation/ dishabituation (i.e. normal levels of interest in novel smells).  No studies to test  learning/memory or motor learning/motor abilities have been conducted on Shank3 mice.  She would welcome input from parents on types of behaviors most typically observed in PMS patients, which would lead to design of specialized studies.

Dr. Crawley listed a huge set of tests done to demonstrate the general health of the Shank3 KO mice. She reported that the mice reached 100% of their developmental milestones, which she used to emphasize that mice are very different from humans.  She also pointed out that the male-female deficits in social interaction are primarily in males. (It should be noted that Shank3 is found in the male gonads.)

Sarah Curran, PhD (King's College London) "Using induced pluripotent stem (iPS) cells to understand the cellular mechanisms underlying SHANK3 microdeletion syndromes"

Dr. Curran focused on a recently started project at the Institute of Psychiatry at King's College London: a new technology to create iPS cell lines by starting from cells derived from a single complete hair.  The goal is to create differentiated cell lines (including neuronal cells) for patients affected by PMS and other ASD syndromes. She is now working with cells from one PMS patient who has an intragenic mutation in Shank3.  The process may take 3-6 weeks from starting with a hair to creating differentiated neuronal cells.  The process is new and needs to be perfected to insure reliability and stability of cell lines before cell lines can be used in further experiments.  Ultimately, if the iPS cells prove to work reliably, this would be a non-invasive method of establishing neuronal cell lines of PMS patients that could be used for testing of potential therapeutic interventions.

Dr. Curran explained that children much prefer the "plucked hair" sample method over skin punches, and that the plucked hair is 100x more efficient for growing iPS cell lines.