Interview #1 – Marisa Bartolomei, Ph.D.
Interview #2 – Huda Zoghbi, MD (PART 1)
Interview#3- Huda Zoghbi, MD (PART 2 & 3)
Interview #4 – Adrian P. Bird, Ph.D.
Interview #5 – Gail Mandel, Ph.D.
Interview #6 – Nat Heintz, Ph.D.
Interview #7 – Stavros Lomvardas, Ph.D.
Interview #8 – Monica Justice, Ph.D.
Interview #9 – Huda Zoghbi, MD (RETT SYNDROME and the DSM V)
Interview #10 – Adrian Bird, PhD [Redefining the Function of the Rett Syndrome Protein]
Interview #11 – Cathleen Lutz, Ph.D. (Jackson Laboratories)
Interview #12 – Jonathan Kipnis, PhD
Interview #13 – Paul Kenny / Anne West / Eric Nestler
Interview #14 – Alysson Muotri, Ph.D.
Interview #15 – Dr. Huda Zoghbi / Jan and Dan Duncan Neurological Research Institute
Interview #16 – Mark Bear, PhD
Interview #17 – Dr. Huda Zoghbi – Rett Syndrome: No Longer Developmental
Interview #18 – Monica Justice, PhD – Bringing Justice to Rett – A Modifier Gene Study
Interview #19- Doris Tulcin – A Mother’s Love Raises the Bar For All Non-Profits
Interview #20 – Bird, Mandel, Greenberg – MECP2 Consortium – A New Way of Doing Business
Interview #21 – Gail Mandel and Dan Lioy – Rett Syndrome – A disease of neurons AND glia
Interview #22 – Hélène Cheval and Jacky Guy – Life Cycles and MeCP2
Interview #23 – David Katz, PhD – Rett Syndrome and Ketamine (Podcast)
April 14, 2009
Marisa Bartolomei, PhD of the University Of Pennsylvania School Of Medicine was awarded funding from RSRT to identify the mechanisms that keep the MECP2 gene silent on the inactive X chromosome.
MC: Dr. Bartolomei, congratulations on receiving funding for this initiative. Please outline for our readership the goals of this project.
MB: The goal of the project is very simple. We seek to identify the kinds of epigenetic modifications that keep this gene off. Everyone now understands what the genome is: all of the genetic material of an organism contained in its DNA. During the last decade or so we have come to appreciate that beyond the genome is the epigenome, which means over and above the genome. The epigenome is changes in gene expression caused by mechanisms other than changes in the underlying DNA sequence. It is these modifications to the MECP2 gene on the inactive X that we seek to identify.
MC: I find the following analogy helpful to explain epigenetics. One can think of DNA as a charm bracelet and epigenetic modification as the charms that can be added or removed to the bracelet.
MB: That’s a good analogy. And it illustrates an important concept about epigenetics: its dynamic quality. Modifications (charms) can be added AND they can be removed. This makes epigenetics quite amenable to drug treatment.
MC: In a best-case scenario, what can you imagine as an outcome of this project?
MB: My hope is that we will identify a specific modification(s) that will, in turn, point to a drug or class of drugs that can reverse the modification. There are a few but nevertheless significant papers which suggest that this could happen. A less encouraging scenario would be if we learned that the modification that keeps MECP2 off is generic and also keeps many other genes off as well. This would be less desirable as we would not want to change the expression of many genes.
MC: Of course having a specific modification would be ideal; however, there are certainly existing drugs, like valproic acid (which many girls with Rett are on for seizures, including my own daughter) that alter the expression of many hundreds of genes. So perhaps, depending on what the genes are, changing their expression may not be so detrimental.
Another concern would be the possible harm done by activating the mutated MECP2 that is silent.
MB: Whether mutated MECP2 does something harmful in the body besides simply not working remains an unanswered question. If mutated MECP2 simply doesn’t work then turning it on should not be harmful. This scenario is referred to a “loss of function”. If, on the other hand, mutated MECP2 does something harmful that extends beyond simply not doing its job (called “gain of function”) then activating mutated MECP2 could exacerbate the situation.
I suspect that some mutations may be simply loss of function while others may be gain of function. This is a key question that the field needs to address.
MC: I learned recently that about 10-15% of genes on the inactive X actually escape silencing and are on.
MB: Yes, that was a rather unexpected phenomenon. We will be able to analyze what is going on in those “escaped genes” and see whether those same mechanisms of escape can be applied to MECP2.
One important question that the X inactivation field is trying to answer is whether the whole X chromosome is turned off and then certain genes are turned back on or if these genes are turned on the entire time. For Rett Syndrome, I think the desirable scenario would be if the entire X chromosome is turned off and then some genes are turned back on. That would indicate to me that the inactive X is not as “locked down” as we may have previously thought and there is room for individual modifications of specific genes.
MC: You have figured out silencing mechanisms of other genes, correct?
MB: I come at this from an imprinting perspective where we have looked at genes where one gene is off and one gene is on depending on which parent it is inherited from. Imprinting is a bit different from X chromosome inactivation where expression is not dictated by the parent but rather is completely random. The random inactivation phenomenon complicates things a bit. Nevertheless, we do have a lot of experience doing this type of investigation with imprinting and we have a lot of experience studying X chromosome inactivation. I feel that the combination of the two fields of research is a very good match for this project. I am looking forward to putting years of experience to good use.
MC: Can you tell our readership how you became interested in this project?
MB: Most of the things that I’ve done in the past have been extremely basic science oriented. It’s very appealing to me to do something that may actually impact a disease. I find that as I get older it becomes more important to me to be able to have that kind of impact. Of course, this project is also intellectually stimulating, which I relish.
Over the years I’ve attended a number of Rett meetings which you have organized, Monica. The more I attend, the more I’ve become engaged with the disorder and felt that my expertise could contribute in a meaningful way to the understanding of this disease.
Through your meetings I’ve had an opportunity to actually meet girls with Rett Syndrome and their families. I believe it’s paramount to bring families and scientists together. We have so much to learn from each other.
MC: Dr. Bartolomei, I thank you for your time and for your interest in Rett Syndrome. I look forward to keeping our readers updated on your progress. I’m sure they join me in wishing you Godspeed!
December 20, 2008
Your discovery in 1999 that mutations in MECP2 cause Rett Syndrome was a breakthrough decades in the making. Can you bring us back to the summer of 1999 and share how the discovery came about?
We knew the general area on the X chromosome where the Rett Syndrome gene resided, an area called Xq28, and we had been searching using logic and a candidate gene approach, systematically analyzing genes involved in neuronal function. One day Ignatia Van den Veyer, a senior person in my lab, was pursuing a new project looking at imprinting and methylation status. I gave her 40 different DNA samples as controls, including a sample from a patient with Rett Syndrome, which I threw in as an afterthought. She found a methylation defect in that one Rett sample. All the others were normal. We went back to Xq28 to look for genes that may have something to do with methylation. That reasoning brought us to MECP2. Interestingly, we had already considered MECP2 but had discarded it as a viable candidate gene because, at the time, it was thought that the knock out mouse models died during embryogenesis. Nevertheless I asked Ruthie Amir, a post-doc in the lab, to sequence MECP2 - just in case. It was the end of August and I had gone to Lebanon to visit my family. I arrived back on a Friday and the phone was ringing off the hook. Ruthie wanted to catch me the minute I arrived. She said, “I think I have a mutation in MECP2.” She came right over and we pored over the data. I asked her to sequence a few more patients and the results were convincing. We wrote the paper in 2 days.
Incredibly, we now know that there are no methylation problems in Rett Syndrome. Furthermore, that one Rett sample with the methylation problems of unknown origin, to this day, does not have an MECP2 mutation. This is a perfect example of how science can be full of surprises and serendipity.
Not every case of clinically diagnosed Rett Syndrome has an MECP2 mutation/deletion confirmation. Do you still feel strongly that Rett Syndrome is a monogenic disease?
If you consider classic Rett with all its particular features we find mutations/deletion in MECP2 in 96% of the cases. The other 4% may include patients who have mutations that are hard to detect. This is not unusual in a gene that is quite large with many regulatory regions. Or they may include patients that present like Rett but have mutations in other genes. It may be that these genes are in the same pathway as MECP2. Fortunately technologies are advancing at such a wonderful pace that sequencing large chunks of DNA is becoming very feasible. I think within the next few years we will be able to test if some of the 4% have regulatory mutations in MECP2 or have mutations in other genes.
Your MECP2 discovery has converged the fields of epigenetics and neurobiology. We think of MECP2 as a master gene controlling many other genes. Tell us why the field of epigenetics is so exciting.
I think the field is very exciting because it is the link between our genome and our environment. If you have a genetic defect in the genome, that is a rather inflexible situation. But the epigenome is much more flexible and can be modulated in response to environment, training and experience. The fact that we now know that epigenetics is so critical for brain function and integrity and the fact that, in time, there will be epigenetic pharmacological agents gives me hope that one day we will be able to modulate brain function.
Your lab has made key contributions in the understanding of Rett Syndrome since the gene discovery. Please elaborate on some of these contributions.
Our lab has worked to understand a variety of things. They can be roughly classified into four areas: clinical research by studying patients; generation and characterization of mouse models; probing the pathogenic mechanisms of the disease, and linking the features of Rett Syndrome to specific neuronal subtypes and gene expression changes.
More specifically we have studied, for example, how different mutations affect the severity of disease. We have looked at the role of X chromosome inactivation and determined that even a small percentage of cells expressing this defect can cause symptoms. We’ve made animal models and have extensively characterized them so that we can detect symptoms of autism and anxiety. We have also studied the phenomenon of synaptic plasticity.
Perhaps a more sobering finding happened when we learned that doubling the protein causes a progressive disorder. This led us to patients and the MECP2 duplication syndrome. We now know that doubling as well as losing the protein is detrimental to the brain. Having animal models has allowed us to uncover some of the mechanism contributing to disease and given us insight into the physiological and neuroanatomical defects.
The finding that both loss and gain of this protein cause synaptic change quite early on points to a developmental component. The brain, being quite plastic and flexible, has the ability, however, to partially compensate so such defects are not always apparent if one looks in adult animals. Having the mouse models allows us to uncover gene expression changes that result from the absence of this protein. The finding that the levels of many genes are altered in a Rett mouse and that many are down in the hypothalamus was a big surprise.
In your opinion what are the most promising areas for future treatment strategies?
Given that the target genes of MeCP2 are many I think it is unlikely that we can utilize them individually as therapeutic targets. A more attractive scenario is to explore the other proteins in the MeCP2 pathway. Targeting those proteins with pharmacological agents may provide therapeutic benefit.
Modifier genes are also an attractive approach. We know from the clinic that there are patients with classic MECP2 mutations and normal X inactivation patterns who do not have Rett Syndrome. In fact, I have an eight-year-old patient who speaks and understand three languages. It is likely that mutations in other genes are protecting this child from developing Rett. Our lab is utilizing two parallel approaches to identify these modifier genes. Using a candidate gene approach in collaboration with Juan Botas we have identified modifiers of MECP2 toxicity in an overexpression model in Drosophila (fruit fly). We now plan to study these modifiers in mammalian cells to test if they are involved in the MECP2 pathway.
A parallel approach is being employed in collaboration with Monica Justice at Baylor. She is utilizing an unbiased mutagenesis screen to identify genes that modify the effect of MeCP2 dysfunction. Mice with random mutations in other genes are bred with Rett mice. A certain percentage of their offspring will have no Mecp2 along with a mutation in some other gene. Dr. Justice will look for mice which have symptoms that are either milder or more severe than is typical for the Rett mouse. She will then track down these mutations in the hope that some will turn out to encode proteins that might make up for the deficiency of Mecp2. This project is progressing well.
What is your lab currently focusing on?
We are studying the function of MeCP2 in selective neurons to uncover features that would be masked if we looked at the whole brain at once. For example, a recent study showed us that MECP2 in the hypothalamus is crucial for dealing with new social situations, for regulating feeding behavior and for responding to stress. Having this knowledge will allow us to begin drug trials in mice to test therapies that hopefully can improve some of the symptoms and bring relief to the patients and their families. We will use this approach to knock out Mecp2 in other parts of the brain.
January 7, 2009
The goal of the institute is to advance our understanding and management of neurological childhood diseases through research using a multi-disciplinary approach. It’s unique because it’s the first time that investigators from vastly diverse fields are brought together in one place to focus on solving childhood neurological disease. The investigators will include physicists, mathematicians, engineers, animal psychologists, and pharmacologists, in addition to neurobiologists, geneticists, and biochemists. These individuals will be working together to understand these devastating diseases. Investigators at the NRI will have state of the art core facilities, for imaging, in vivo physiology and behavioral analysis. The NRI has been designed based on years of experience and knowledge of the bottlenecks that hinder research.
What kind of resources will be directed towards Rett Syndrome research?
The NRI will have a large operational budget to sustain the kind of infrastructure that is required to run interventional drug trials in parallel. For example, the animal facility will be able to house 80,000 mice. And there will be multiple rooms dedicated to behavioral studies to determine if therapies are beneficial. We’ve decided that we should focus on a handful of diseases: Rett Syndrome, Angelman, Fragile X, cerebral palsy caused by periventricular leukomalacia due to prematurity, and autism. We hope that by making significant strides with these diseases we will have proof of principle that our philosophy at NRI works and we can, in time, move beyond these initial diseases.
When do you expect the Institute to be fully operational?
We expect NRI to open its doors fall of 2010 and additional faculty will continue to be added through 2011. NRI will include 15 -18 faculty members, each of whom will have their own independent laboratory. As we have already discussed there will also be core facilities, each run by a core director.
Part Three – Rett Syndrome and autism
January 7, 2009
Can you elaborate on what clinical features Rett and autism share? How do they differ?
Both disorders are characterized by loss of language, stereotypies, problems with social interactions. As girls with Rett age their social interactions typically improve but they continue to engage in stereotypies and to lack language skills. There is a subset of girls who never lose eye contact. There is no doubt however that when Rett sets in there is a change in the social behavior of the child that is very reminiscent of autism. Also the timing of the postnatal onset of the regression is similar in both disorders, typically around 18 months of age. Other symptoms in common include gastrointestinal problems and seizures.
The most obvious difference is the severity of physical challenges that many children with Rett Syndrome suffer; that is not seen in autism. Also, as children with Rett grow we see more social engagement with the environment than is typically seen in autism.
From a molecular perspective how are they related?
We know that from a molecular perspective autism and Rett are definitely related. We have patients that have a diagnosis of classic autism (these are not Rett patients nor are they atypical Rett) that have mutations in the MECP2 gene. In fact, mutations in MECP2 account for 1 – 3 % of all cases of autism.
Currently, Rett Syndrome is classified as an autism spectrum disorder. Not everyone agrees with this classification. What is your opinion?
This is a complicated question but it’s imperative that we handle this situation thoughtfully. Some people feel that having a known genetic cause should be reason enough to reclassify a disorder. For Rett and autism the case is more complicated. There are patients with classic autism that have no features of Rett but have MECP2 mutations. They have one diagnosis and one diagnosis only: autism. You cannot just blankly declassify Rett and MECP2 disorders from autism spectrum disorders. At the very minimum you have to include MECP2 spectrum disorders under the autism spectrum disorders umbrella.
The argument that because the genetic defect is known we have to remove Rett is also not a good argument. In 10 years we will know more and more causative genes for autism. Hundreds of genes will be identified as causing autism – will all those patients be removed from the autism spectrum disorder classification? Where will that leave them?
December 20, 2008
It’s been 1 ½ years since your reversal paper was published. How did the original mice fare post-publication?
Mice normally live for a couple of years. And it was no different for our reversed mice. In other words, reversal of both male and female symptoms was very effective indeed.
Can you elaborate on efforts to confirm the results in either your lab or other labs?
We have done the reversal many times and have no doubt that the results are robust. The outside world however needs to see that other groups can reproduce our experimental findings. A group from Novartis has verbally presented results which confirm our findings using related, but not identical, experimental protocols. This work has not been published yet.
Have you performed any analyses on the mice that were not included in the original paper?
We have done some but not as much as we had hoped because of an infection in the mouse house that needed to be eradicated. For example, we have done an analysis of arrhythmic breathing and find that in both males and females this is fully reversed. We are in the process of generating enough mice to do in-depth behavioral tests of other kinds and also to look at nerve cells more closely to see how well they have recovered their structure. We are encouraged that the symptoms we have analyzed to date have all been reversed.
What are the next steps underway in your lab that follow up from the reversal?
We are looking at ways of either switching on or off the MECP2 gene in specific regions of the brain in order to pin down where symptoms of Rett Syndrome originate. Parallel efforts in other labs including Lisa Monteggia (UT Southwestern) and Huda Zoghbi (Baylor College of Medicine) have reported experiments of this kind which look promising and we hope to contribute further information on this issue. In addition we are collaborating with Stuart Cobb (University of Glasgow) and Gernot Reidel (Aberdeen University) to study the physiology of both MECP2 deficient and “reversed” nerve cells in the mouse brain. (Click here to learn more about this RSRT-funded project.)
Besides the reversal follow-up what other Rett research is your lab working on?
We are trying to better understand exactly what MECP2 does in nerve cells. MECP2 function is controversial as several ideas are in play. We are making quite good progress and hope to have a clearer view of MECP2 function in the near future.
Given the extent of systems that are dysfunctional in Rett it is surprising that it is reversible. What is your best guess regarding what MECP2 is doing in the brain and how can restoring it cause severe symptoms to be reversed?
The best available evidence indicates that MECP2 binds to DNA in the chromosomes due to chemical marks known as DNA methylation. We suspect that when MECP2 is missing the pattern of DNA methylation is unaffected. This means that restored MECP2 can go to its chromosomal sites as dictated by the pattern of methylation and take up its normal function. We are not sure if this is the correct explanation and that is why further experiments are underway to test this idea. With hindsight we were perhaps more surprised by the reversal than we need have been. It is assumed that neurological disorders of this kind are irreversible even though very few tests of this idea have ever been performed. New data from other diseases such as Fragile X Syndrome and tuberous sclerosis also point to a significant degree of reversibility even though these disorders were also thought to be irrevocable. Overall, I think the new data is causing a reappraisal of everybody’s view of these disorders.
What impact has the reversal had on the Rett research field?
It has opened a lot of doors in research terms but it’s still early days to see the full impact. We now have proof of concept that Rett Syndrome is likely to be a curable disorder. That changes everybody’s perception about how to move forward – in a hopeful way.
What are your thoughts on areas that hold the most promise for therapeutic intervention?
Any potential therapeutic approach can now be entertained. Just because we used expression of the MECP2 gene to reverse symptoms in mice does not mean that gene therapy or a similar protocol has to be the preferred approach in patients. In other words, we need to try drugs in both a rational and blind screening approach, we need to look at the possibility of activating the silent MECP2 on the inactive X chromosome and we need to look at influencing other genes that can reduce the severity of Rett symptoms. Quite honestly, at this stage it’s not possible to bet on one particular approach and indeed it may be that combined therapies will eventually be most effective.
(Visit the RSRT website for more information on this topic.)
Extra: Listen to a BioPod interview of Professor Bird regarding the reversal experiment.