A paper just out in PLOS Biology is unable to confirm that the drug Ataluren is able to read through premature stop codons (nonsense mutations, for example 255X, 168X). The drug is already in clinical trials for Cystic Fibrosis and Duchenne’s  muscular dystrophy.   The new data highlights not only the difficulties in drug development and the importance of rigorous clinical trials but also the need for independent confirmation of data.  This drug is currently being tested in a mouse model of Rett Syndrome with a 168X mutation.

Drug Mechanism Questioned
A study fails to confirm that the small molecule PTC124, in development for multiple genetic disorders, aids in read-through of premature stop codons.
By Kate Yandell

Assays using glowing luciferase from fireflies play a controversial role in PTC124's history.

Assays using glowing luciferase from fireflies play a controversial role in PTC124’s history.

An in vitro study of PTC124, a drug in clinical trials for the treatment of cystic fibrosis and several other genetic disorders, has some scientists wondering whether the molecule works the way its developers say it does.

PTC124 is intended to treat disorders, including some cases of cystic fibrosis and Duchenne muscular dystrophy, that stem from mutations that cause stop codons to erroneously appear in critical gene transcripts, thereby inhibiting translation of vital proteins. The new study, published today (June 25) in PLOS Biology, demonstrates that PTC124 does not promote read-through of such premature stop codons, or “nonsense” mutations, in several reporter assays done using a human cell line.

“The paper is well designed and carefully executed, and the dataset is unambiguous,” said Bryan Roth, a professor of pharmacology at the University of North Carolina at Chapel Hill who was not involved in the work, in an email to The Scientist. “I think these are important findings which cast doubt on the role of nonsense suppression on the actions of PTC124.”

“From a basic mechanistic standpoint, it doesn’t look like [PTC124 is causing] translational read-through, certainly in our assays,” said Stuart McElroy, a molecular pharmacologist at the University of Dundee in Scotland and an author of the paper.

But representatives from PTC Therapeutics, the New Jersey biotech company that is developing the drug, said that it has produced strong evidence that PTC124, also called ataluren, does encourage the translation of complete proteins despite the nonsense mutations. “Numerous independent laboratories have provided confirmation of our results, demonstrating ataluren’s read-through activity in studies using reporters as well as multiple animal and cell-based nonsense mutation disease models,” the company said in an emailed response to questions sent by The Scientist.

Read More …

Press Release Announces Tim’s Hire

Dear Friends,

Tim-and-Eleanor-2A year and a half ago my wife, Rachel, and I received the worst phone call of our lives—it was the Children’s Hospital of Pennsylvania informing us that our beautiful, bright-eyed, giggling two-and-a-half year old daughter, Eleanor, had tested positive for the MECP2 mutation that causes Rett Syndrome.  We were simply devastated and didn’t know what to do or where to turn.  The ensuing months were the hardest of our lives.  Our dreams and hopes for our only child had been crushed.  We watched helplessly as Eleanor stopped scooting on her rear end, as she had been doing, and developed a repetitive hand motion and other symptoms.

Several months later, Rachel found the RSRT website.  We made a call and RSRT’s co-founder and executive director, Monica Coenraads, answered the phone.  We soon learned that we were not alone in our sadness and that there was a community of wonderful, supportive parents, grandparents, family members, and friends of families who have been impacted by Rett Syndrome.  And even more exciting, we learned that there was hope for a cure for Rett.  In fact, it was clear that there was a good deal more than hope—Rett symptoms had been reversed in an animal model, and very promising scientific progress was being made, much of it encouraged and supported by RSRT.

Monica and I continued to correspond.  I was seeking her advice and thoughts about Eleanor’s diagnosis, and I was trying to understand the research and science.  Monica began seeking my advice about fundraising and public relations when she learned that I headed the development office of the Woodrow Wilson Foundation in Princeton, New Jersey, and before that had served as director of development at Columbia University’s Teachers College.  It may have dawned on Monica and me at the same time that there was a fit here—that I cared deeply and personally about the work that RSRT was doing and that I had nearly 20 years of experience that could help grow RSRT and its impact on the lives of girls and women with Rett.

Tim-and-EleanorThe rest, to use the cliché, is history.  My first official day as a Program Director at RSRT was June 17.  Under Monica’s direction, I’m responsible for fundraising, public relations, and strategic thinking about the organization.  I couldn’t be more excited.  So many people, Monica foremost among them, have worked so hard and contributed so much to making RSRT the respected force that it is and to building the cumulative scientific knowledge that will lead to a cure.  I’m honored and humbled to join this team.  Frankly, I never imagined that I would be able to put my knowledge and skills to use for something so important to me.

I’ve gone on longer than I intended, but I have one further thought.  I’ve been thinking lately about President Kennedy’s 1961 speech to Congress in which he announce the dramatic and ambitious goal of sending an American to the Moon before the end of the decade.  It took a huge team effort, conviction and confidence in a very clear mission, ample resources, and leadership, for the goal to be met.  I think RSRT’s goal is not unlike President Kennedy’s in its ambition, its clarity, its importance, and its attainability.  I also believe that all of us working together are the team that will get us to the moon.  And, like building a rocket, a cure depends on cumulative knowledge that is the sum of its parts.  The rocket needs its nose cone, its fuel tank, its electronics, its landing pads, and other components to meet the goal.  Research is like this too.  All the parts have to work together.  It is cumulative knowledge that will get us there.

Tim-and-Eleanor-3I am tremendously grateful to Monica, to the RSRT Board, and to all of you who contribute your time, energy, and resources to RSRT for your confidence in me.  I promise Eleanor and all of our daughters that I will do my best in everything I do for RSRT.  I will need your help, advice, and counsel—most of you know RSRT and all of its accomplishments and the Rett community at large far better than I do—so I hope I can call on you.  Please don’t hesitate to contact me any time.  My office line is 609-309-5676; my cell is 609-815-5102; and my email is tim@rsrt.org.  I look forward to meeting you.

Best,

Tim Freeman

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Adrian Bird (left) and Matt Lyst
University of Edinburgh

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Michael Greenberg (right) and Dan Ebert
Harvard Medical School

It stands to reason that in our battle to cure Rett Syndrome it would be of great benefit to understand the function of the “Rett protein”, MeCP2. Towards this end RSRT launched the MECP2 Consortium in 2011, a unique $1.8 MM collaboration between three distinguished scientists, Adrian Bird, Michael Greenberg, Gail Mandel.  On June 16th the first two publications from this collaborative effort are published in Nature Neuroscience and Nature. Together these papers provide further clarification of the elusive function of the MeCP2 protein and how mutations within it contribute to Rett.

We thank Kathy and Tony Schoener whose visionary $1 MM gift made the Consortium possible. We thank all of our donors and parent organizations worldwide who support us, in particular our funding partners Rett Syndrome Research Trust UK and the Rett Syndrome Research & Treatment Foundation.

We are providing a variety of resources to help you understand the progress being reported today.

Press release

Animation of Nature Neuroscience Paper (courtesy of Jeff Canavan)

Chinese Translation

Interview with Matt Lyst, post-doc in Bird lab

Interview with Michael Greenberg and Dan Ebert,
post-doc in Greenberg lab

We break down a few interesting results recently published.

Multiple mutations on MeCP2

In most cases of Rett syndrome, one mutation occurs on (a single copy of) the MeCP2 gene. But, rarely, multiple mutations occur on the same copy. Researchers from the University of Alabama at Birmingham characterized the largest group of individuals to date, 15 of them, who have more than one mutation. (The girls were participating in the Rett Syndrome Natural History Study, which aims to gather detailed historical and physical examination data on a large cohort of females with Rett — of which there are now more than 800.)

In contrast to two previous case studies suggesting that girls with more than one MeCP2 tend to have more severe disease, the new study found that participants with multiple mutations assessed using two quantitative measures — the Clinical Severity Scale and the motor behavioral analysis — showed no difference from those individuals with single, similar mutations. The findings appear in the American Journal of Medical Genetics Part A.

Other factors, including the individual mutations themselves or how much of the mutated gene is inactivated as part of normal development, can worsen symptoms, the authors suggest. Future drug therapies that account for a person’s genetics should consider this small group of individuals, the authors add.

Rett Syndrome and epilepsy

Seizures affect 50% to 90% of individuals with Rett syndrome. According to a recent review in Pediatric Neurology by Alison Dolce and her colleagues at Johns Hopkins Hospital in Baltimore, Maryland:

  1. It’s unclear whether earlier onset of seizures signals a poorer overall prognosis.
  2. EEG, or electroencephalography, an indirect measure of brain activity, is an important diagnostic tool in children with Rett syndrome because it allows clinicians to identify true seizures — as opposed to symptoms, ranging from hyperventilation or motor abnormalities, that are often mistaken for seizures. From 2 to 4 years of age and beyond, all girls with Rett syndrome develop abnormal EEGs, the authors write.
  3. There are few studies assessing anticonvulsant therapies in Rett, and many of them are short-term and involve only a small number of participants. “At our centers, we have circumstantial evidence of good responses to levetiracetam, lamotrigine, and topiramate,” the authors write. Although the clinicians also use valproate, the side effects can be problematic. In general, many children with Rett require more than one medicine.
  4. It’s still difficult to predict which patients will tend to develop seizures early and also whose seizures will be easier to treat.
  5. According to the authors, physicians should begin to consider the ketogenic diet and vagal nerve stimulation earlier in the clinical course of Rett, because these treatments “might be helpful.”

More on MeCP2 mechanisms

MeCP2 protein has several defined parts that researchers are studying. The methyl-CpG-binding domain or MBD, for example, binds to DNA in studies conducted in vitro and is thought of as crucial for the protein’s function of turning genes on or off, or altering the way DNA is stored in a cell. Given that the interactions between MeCP2 and DNA are difficult to mimic in a dish, a team of Canadian scientists wanted to see what would happen to the interaction in a mouse whose front end of the protein was deleted. The Mecp2tm1.1Jae model, commonly used in studies of Rett syndrome, is missing its first 116 amino acids—which include the 48 amino acids that make up the MBD. (What’s more, MeCP2 mutations in people with Rett occur in this region, such as R106W, R1333C, P152R, F155S and T158M.)

In the study, mutant mice still had MeCP2 in their tissues but only about half as much as healthy mice did. Although the mutated MeCP2 still bound to DNA and chromatin, the interactions were weaker and less specific for methylated DNA. In addition, although the ‘nuclear localization signal’ region of the mutated protein was still intact, protein was less able to move from the cytoplasm to the nucleus, where it can influence the production of other proteins. The paper is published in the May issue of Nucleic Acids Research.

News from the Fragile X community highlights the challenges of clinical trials.

Below is an article from the New York Times written by Andrew Pollack.

FRAGILE-articleLarge

Holly Usrey-Roos will never forget when her son, Parker, then 10, accidentally broke a drinking glass and said, “I’m sorry, Mom. I love you.”

It was the first time she had ever heard her son say he loved her — or say much of anything for that matter. Parker, now 14, has fragile X syndrome, which causes intellectual disability and autistic behavior.

Ms. Usrey-Roos is certain that Parker’s new verbal ability resulted from an experimental drug he was taking in a clinical trial, and has continued to take for three years since then. She said she no longer had to wear sweaters to cover up the bruises on her arms she used to get from Parker hitting or biting her.

Now, however, the drug is being taken away. It has not met the goals set for it in clinical trials testing it as a treatment for either autism or fragile X syndrome. And Seaside Therapeutics, the company developing it, is running out of money and says it can no longer afford to supply the drug to former participants in its trials.

The setback is a blow in the effort to treat autism since the drug, arbaclofen, was one of the furthest along in clinical trials. And the company’s decision has caused both heartbreak and outrage among some parents.

“I waited 10 and a half years for him to tell me he loved me,” said Ms. Usrey-Roos, who lives in Canton, Ill. “With fragile X, you’re like living in a box and someone is holding the lid down. The medication opened the lid and let Parker out.”

“I don’t want to go back to the way life was,” she added.

The situation raises questions about what, if anything, drug companies owe to patients participating in their clinical trials. It also points out the difficulties in developing drugs to treat autism and fragile X syndrome. If the drug worked so well in some patients, why has it not succeeded so far in clinical trials?

Read the full article …

hope-pic

Guest blogger, Beth Johnsson, with her daughter Hannah.

by Beth Johnsson

Someone once told me that hope is what distinguishes humans from every other species; our ability to look to a potential future rather than live solely in the here and now. This is, of course, wildly inaccurate; I am not a biologist or anthropologist (or any other ‘ist’ for that matter), but I believe there are a number of other factors which separate humans from the species around us (not least, an opposable digit!). But the concept has stuck with me and, since most of my life is built around hope, it cannot help but strike a chord.

Three weeks ago, Hannah had four medical appointments in five days and a multitude of other issues which needed addressing. Hope permeated them all. I hoped, for example, that we would manage to get a disabled parking space at the hospital; I hoped that the wait wouldn’t be so long that we lost Hannah’s good will before we even got in the room; I hoped that the ECG results would show that the recent episodes we have observed are not seizures; I hoped that the locum SLT (who barely knows Hannah) would recognize that eye gaze is her best possible chance at communication and would therefore support our application for the technology; I hoped that the engineer would say he could adapt Hannah’s trike to make it big enough for a child who should be riding a bike; I hoped that the physio would approve a height adjustable bed to prevent our inevitable back injuries; I hoped the eye unit would finally discharge us because, (take note SLT!), her eyes do work; I hoped the blood tests results would require no more than three adults to hold Hannah down; most of all I hoped we would make it through each appointment without a total meltdown (Hannah’s, not mine!)

Sorry, that’s wrong, that is not what I hoped most of all. Most of all, I hoped, with every second of every appointment and of every minute in between, that one day none of this will be needed. I hoped that one day the ‘professionals’ involved in my little girl’s life will be her teachers, her GP and her dentist. And that’s all.

Two weeks ago, the routine medical appointments were replaced by an unexpected admission to hospital. Her first. (What a strange world we now live in, where I know that making it through six years without a hospital admission makes us incredibly lucky). An infection, the source of which remains unknown, has made my cheeky and (hitherto) still mobile little girl, lethargic, disengaged, and unwilling to walk. Most worryingly, it has put out the sparkle in her eyes. Now the professionals involved reach an all-time high, as does my reliance on hope. I watch her suddenly unable to take a step on her own, trembling violently, and I cling fiercely, with muscles I didn’t even know I have, to the hope that this is not the beginning of regression, the start of a life time spent in hospital, but ‘just’ the temporary result of an infection. Something they do have a cure for.

When Hannah was first diagnosed I didn’t know enough (and didn’t allow myself to know enough) about Rett Syndrome to understand that a cure was the only hope. Then, once I started to learn more, I didn’t allow myself to believe that a cure in her lifetime was possible. It seemed too fantastical, too far out of reach, to think it could really happen in time for my little girl. Now, as I continue to learn more about what Rett really means for Hannah, and about the research going on today, I realise that a cure is not only possible, it is the ONLY possible future. I cannot allow myself to think about the possibilities of the alternatives.

Nor can I help but be frustrated and confused by those who don’t seem to share or, at the very least, support that hope. Why do ‘friends’ click ‘like’ on every trivial Facebook message out there, but ignore my posts about Rett? Why can they not take the time to vote when funding is at stake? Why can they not spare the cost of a skinny latte to help make the hope reality? Why do they so often seem to think that I should just accept how things are? Would they? I hope not! It seems to me that to accept is to admit defeat; to hope is to fight. If my hope was false, my fight for an impossible prize, I could understand why acceptance might be healthier, more practical, but it is not false. The prayer I offer every night is not one born of blind faith, for a miraculous thunderbolt from an omniscient being; it is one born of proven fact, for a miraculous breakthrough by a handful of knowledgeable scientists, supported by a group of dedicated parents, in whom my faith lies and on whom my hope depends.

Perhaps others think I should accept because they wonder about the merits of a life based on hope, on a dream for the future. Carpe diem and all that? To be honest, sometimes I have wondered too. Shouldn’t we be living for today, enjoying what is here and now rather than always looking to a future which, ultimately, we cannot guarantee will arrive in time? But the two are not mutually exclusive, surely? In fact, I would say they are co-dependent. Living in hope for the future makes today more positive too: it enables you to notice the tiny, almost imperceptible steps being made forwards, the achievements which others might miss, but which you know are all part of the journey. Why should hoping for a brighter tomorrow preclude you from seeing the light in today? I don’t think it does; the light which research has switched on for Hannah’s future shines in her today too. It illuminates all the other reasons to be hopeful and grateful. When you are in darkness, finding the light switch is hard, so the darkness continues, self-perpetuates, the exit remains elusive. But when you have a little light shining already, no matter how small, finding your way towards the brighter, bigger light in the distance becomes an easier journey.

Before we discovered Rett Syndrome Research Trust and the research they fund, the sense of helplessness was overwhelming. For a control freak, like me, it was impossible. Uncertainty about the future is bad enough, but feeling there is nothing you can do to change it is torture. Even fundraising didn’t feel truly hopeful, when ultimately we knew money was going towards coping with diagnosis, not in making that diagnosis a demon of the past. When I look back on that time, I remember a very dark place, not simply because of the diagnosis itself, but also because of our lack of vision of the future or of how we could influence it. Hopelessness for the future meant helplessness today. The relief that comes from feeling that you are actually doing something, that you are taking action, raising money, helping to fund the science which is holding all your hopes in its hands, this relief is a light switch. Since we turned it on, both tomorrow and today have seemed a great deal brighter.

I started writing this, and thinking about hope, several weeks ago. Every time I think it’s done, something else comes along which is so tightly bound up with hope, some new experience or emotion which makes our hopes shift and metamorphose once more, that I have to start again. When I started writing, Hannah had never been in hospital overnight. She was walking confidently, progressing, even. She’d taken an independent step or two up the stairs. We were daring to hope she might continue. Now things have changed and with them, our hopes. Today we are hoping that she returns to where she was three weeks ago, now just that would be a little miracle. I’m sure all parents’ hopes for their children change over time, evolving inevitably as the child grows and develops their own set of hopes and dreams. I expected that. I just never thought that one morning I would wake up hoping that my six year old will bear her own weight. Everything is relative – our daily, weekly, monthly hopes change, but the ultimate hope is a constant, one of the few things in my daughter’s life which will not be lost.

I started, all those weeks ago, by talking about what distinguishes humans from other species. Speech, surely has to be one of our greatest gifts. The very thing I hope for most for my daughter. I make a joke of the opposable digit, but the gift the thumb brings to us is the ability to grasp, to hold, to use our hands in ways which other animals cannot. Another fundamental skill my little girl has lost. It’s not that the loss of these things makes my daughter any less of a human being, but I cannot help but believe that it does make her a little less of Hannah: the little girl, teenager, woman she could be. I cannot know if Hannah has hope, whether she is aware enough of the things she cannot do to hope that one day she will, although the way she looks at her brothers playing and running and talking, it is hard to believe that she is not hoping to join them one day. If hope gives her the same sense of purpose and drive and determination as it brings the rest of us, then I hope that she does have hope, and that one of these days my stubborn, cheeky, sparkling little girl will tell me that her name is Hannah.

RETT SYNDROME RESEARCH TRUST WEBSITE

by Kelly Rae Chi

Rett Syndrome doesn’t usually run in the family. Researchers led by Alessandra Renieri at the University of Siena in Italy encountered two exceptional cases: one pair of sisters with the same mutation in the Rett-causing gene MECP2, and a second pair with identical deletions within the gene.

Prof. Alessandra Renieri  University of Siena, Italy

Prof. Alessandra Renieri
University of Siena, Italy

Despite having the same mutations in MECP2, the sisters represented the clinical spectrum of the disorder. For each pair, one sister had classical Rett Syndrome—she was unable to speak or walk or use her hands—while the other had a milder form of the disorder (called Zapella) and could talk using short phrases, walk and retained some hand function. Researchers describe these four women and possible genetic reasons why the severities of their symptoms were so different, in a PLOS ONE paper published a few months ago.

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It’s not surprising that girls with Rett Syndrome generally show a wide range of symptoms. That’s partly because a mutated copy of the MECP2 gene is located on only one of two X chromosomes in a female cell; the other copy is healthy. One X chromosome becomes inactive in each cell early in development. In rare cases when many cells express the healthy copy of MECP2, women show a milder form of Rett. In the new study, however, both pairs of women were similar in how many of their maternally or paternally derived X chromosomes were inactivated, suggesting that something else might explain the differing severities of their disease.

Hypothesizing that other genes could contribute to these differences, the scientists sequenced a small proportion of the women’s genomes (about 1%) that is thought to code for proteins. (This strategy, called exome sequencing, is a less costly and less burdensome in terms of data analysis compared with whole-genome sequencing, and in recent years it has been shown to identify previously unknown genes for rare, inherited disorders, such as Freeman-Sheldon syndrome.)

The team located 112 genetic variants on 108 genes that were exclusive to the women with classical Rett. A subset of these variations, about 10 to 20, is believed to be relevant to impaired protein function in Rett, based on what’s already known about them.

These genes are involved in a range of functions. Interestingly, both women with classic Rett have variants on at least six genes that have been previously linked to oxidative stress. (The two people with Zappella had variants on three.) In a follow-up study, Renieri’s group found that the women with classical Rett, but not the two with Zappella, showed molecular signs of oxidative stress compared with healthy controls. But the link between MECP2 mutations and oxidative stress is still unknown, the authors note.

The women with Zappella had exclusive variants in 80 genes, but none of these were shared by both. Some genes are linked to immune function, and the variants may be involved in protection from a more severe phenotype, says Renieri, a professor of medical genetics.

Although exome sequencing will continue to bear genetic clues on the variability of Rett Syndrome, the meaning of these variants will need further study, Renieri says. “I’m not sure now that all the variants we describe in the paper are relevant,” she admits. “In the next few years we will learn better how to interpret these results.”

Renieri’s group hopes to sequence the exomes of more people with severe and mild Rett Syndrome, to understand their genetic similarities and differences. It is easier to compare the genes of sisters because their genomes are 50% identical. But because sisters with Rett are so rare, they will need to compare unrelated patients, she says.

RETT SYNDROME RESEARCH TRUST WEBSITE

On April 23rd in New York City RSRT presented an event entitled “Curing Rett Syndrome – How Do We Get There?” The event was videotaped and is now available on RSRT’s YouTube channel.

Monica Coenraads
Executive Director of RSRT

Curing Rett Syndrome – How Do We Get There?

Monica gives an overview of the various approaches from gene therapy to drug screens and everything in between that are being pursued to cure Rett Syndrome. We encourage you to invest 30 minutes of your time and arm yourselves with the facts.

VIDEO TRANSCRIPT

Italian Translation

Spanish Translation

German Translation

Chinese Translation

Korean Translation

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Ben Philpot, Ph.D.
University of North Carolina at Chapel Hill

Gene Awakenings for the Treatment of Neurological Disorders

Late last year RSRT made a $2.2 million investment in the labs of Ben Philpot and his colleagues to conduct a screen to identify compounds that can unsilence a healthy copy of the mutated Rett gene. The screen is now up and running. Learn the basics of how the screen works and why we are optimistic about this approach.

VIDEO TRANSCRIPT

Chinese Translation

RETT SYNDROME RESEARCH TRUST WEBSITE

independent

johnsson

The following piece comes to us from the blog of a UK newspaper, The Independent. This powerful and poignant piece was written by Beth Whitley mother to Hannah who has Rett Syndrome.  (3/15/2013)

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Life with Rett Syndrome: ‘When my little girl was diagnosed, I had no concept how much things were going to change’

by Beth Whitley

I lost an old friend this week. Not in the idiomatic sense that he passed away, nor in the literal sense that I misplaced him in a crowded supermarket and never found my way back to him. Although, metaphorically, perhaps that’s exactly what happened: we lost each other in the crowded supermarket of life and by the time we realised we’d gone astray, there were just too many aisles and trolleys and shelves of tinned goods to find our way back. Of course, if I hadn’t been pushing a wheelchair maybe I’d have been able to keep up a bit better.

[continue reading]

RETT SYNDROME RESEARCH TRUST WEBSITE

by Kelly Rae Chi

Rett Syndrome is caused by a variety of mutations in the MeCP2 protein, but in some instances, MeCP2’s end is missing.  A graduate student in Developmental Biology at the Baylor College of Medicine in Houston, Steven Baker, who is also in the medical scientist training program, was sifting through the clinical literature on boys with such mutations when he noticed that a tiny difference in how much of the protein’s tail is shortened—by just three amino acids—seemed to make the difference between decades of life (albeit with Rett-like deficits) and death in infancy.

Steven Baker

Steven Baker

Baker asked his adviser, Huda Zoghbi, whether she thought those extra few amino acids could so dramatically change the clinical progression of Rett.

Huda Zoghbi

Huda Zoghbi

“I don’t know,” Zoghbi, Howard Hughes Medical Institute Investigator and director of the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, recalls telling him, because other genetic factors could be contributing the dramatic difference in the progression of Rett. “The only way to know is to make mice that have the two different mutations.”

So they did. One mouse had the end of its MeCP2 cut off at the 270 mark (‘R270X’ mice) while another’s protein was shortened at the 273 mark (‘G273X’ mice). The stories of these mice were reminiscent of the boys Baker had noticed. The R270X mice died prematurely, around the same time as mice with no MeCP2. In contrast, the G273X mice, with their extra three amino acids, survived longer and showed symptoms later, although these features became more severe and the mice died before healthy mice did.

What do those amino acids do? In trying to find out, the scientists have refined their understanding of how MeCP2 works. Their results are published this week in Cell.

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A first look at DNA binding:

Researchers know that MeCP2 binds throughout the genome, coating the DNA in neurons more heavily than in other cells, but what exactly it does after that is less clear. It’s thought to turn genes on or off, or alter the overall structure of DNA.

Zoghbi’s team thought that the two truncated forms of MeCP2 might bind to DNA differently — in a way that would help explain the different clinical progressions of the boys — but when they initially looked at several spots within the genome they saw that both forms bound to those spots similarly.

In fact, looking more broadly across the genome the group found that overall binding of the MeCP2 was pretty much the same, and it looked normal. (The latter wasn’t too surprising, though, because the front end of MeCP2 was already known to bind to DNA.) Both mutations also interfered with the normal ability of MeCP2 to repress genes.

Looking more closely at gene expression at various time points in brain development, however, the group found a key difference in the two mutants: at 4 weeks of age, a small group of genes was improperly expressed in the R270X mutant but not in the G273X mutant. Interestingly, most of those genes eventually became misregulated in the G273X group by 9 weeks.

The hook from the animal kingdom:

Meanwhile, the scientists asked evolutionary biologist, Olivier Lichtarge, a Professor at Baylor College of Medicine who uses computational tools to study the evolution of protein sequences, to help them compare the sequences of the protein’s tail across different species (the idea being that any regions of the protein that were highly similar across species might be important for the protein’s function). “We worked with Angela Wilkins in the Lichtarge lab and asked, ‘Is there something in this domain that’s really unique?” says Zoghbi, who is also a Professor at Baylor College of Medicine.

They found that three clusters of MeCP2’s tail were highly conserved across fish, frog, rat, mouse, and human. Shortening MeCP2 at the 273 mark removed the third of those conserved clusters, whereas cutting the tail at the 270 mark deleted the second and third clusters.

What are these clusters in MeCP2’s tail and what do they do? It turns out that they’re called AT-hooks. In 2005, a study by Adrian Bird’s group described the first of those three AT-hooks (which is the one still present in both of Zoghbi’s new models), though its function was unclear.

AT-hooks are regions of a protein that are already well known to bind DNA, however, so the team went back to the idea that the two truncated proteins might differ from one another in how they attached to the genome, even though their initial results had shown that binding was similar. Using a different assay, they found that missing the second AT-hook domain impaired the ability of the R270X to steadily interact with certain sequences in the genome.

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The interaction between MeCP2 and DNA:

Our genomes are wound tightly around spool-like proteins called histones; the DNA and histones (together, called chromatin, which looks like beads on a string) are then packed in even more so that it can all fit inside cells. Using an experimental model of compaction in vitro, the team found results suggesting that R270X mice (which, remember, are missing an two AT-hooks instead of one) don’t pack up chromatin as well as 273X mice do.

The initial finding that both mutated forms of MeCP2 bind to DNA are still important though, Zoghbi says. “It tells us that the major binding to DNA happens. That’s the first step.” The researchers think that once the front end of MeCP2 sits on DNA, the AT-hook clusters on its tail come in manipulate the DNA further, likely bending or altering the structure to help it pack further into the cell.

“There were hints previously that MeCP2 might cause a change in the overall conformation of DNA. The new study is probably the most direct evidence,” says Howard Hughes Medical Institute Investigator Gail Mandel, at the Oregon Health and Science University in Portland, who was not involved with the study.

The protein partner ATRX:

When the MeCP2 sits on DNA, and likely alters the way it packs into a cell, there are other molecular partners that come and join it. One of those is the protein ATRX—whose mutations have been linked to Alpha-thalassemia mental retardation syndrome—and Adrian Bird’s lab has previously shown that its interaction with DNA is disrupted in mice missing MeCP2. Zoghbi’s team decided to look at this protein in their new mutant mice.

Compared with the healthy mice and the G273X mice, ATRX goes missing from the tightly packed DNA of neurons earlier in life for the R270X mouse, and this loss mirrors the quicker onset of Rett symptoms. “To us, that was really interesting,” Zoghbi says, “because this change is not because the neurons are sick, it’s because you don’t have MeCP2 functioning properly.”

Studying female mutant mice that are missing a copy of MeCP2, the researchers found those brain cells with no MeCP2 also had less ATRX bound to tightly packed DNA compared to controls. In female mutants with too much MeCP2, an excess of ATRX latched on to DNA. MeCP2’s absence from liver and non-brain organs didn’t affect ATRX binding in those organs, suggesting that MeCP2 has a mechanism that’s specific for the brain.

Future directions:

Taken together, these results suggest that in the brain, the AT-hook clusters on MeCP2’s tail are manipulating DNA in a way that’s crucial for the other protein partners to bind and do their jobs, Zoghbi says.

“This new paper is beginning to shed light on the complexity of this interaction between MeCP2 and ATRX,” says Mandel. In addition, “we don’t know all the other proteins that bind to MeCP2, but the guess would be that there are likely more partners affecting whether genes are on or off.”

Zoghbi’s team hopes to understand how shortening MeCP2’s tail changed chromatin structure without dramatically changing gene expression — as well as the mutation’s affect on brain activity. They also plan to do biochemical and molecular experiments to figure out where ATRX is going and what it’s doing when its distribution is altered in the brain cells of the MeCP2 mutants.

For Zoghbi, the new findings underscore the importance of going back to patients to look for clues about MeCP2’s function. In 1999, Zoghbi first showed that various mutations in MeCP2 caused Rett. “Here we are 14 years later, some of these human mutations are teaching us lessons,” she says. “The variety you get and the breadth of human features you can dissect and go back and study in the mouse are really very humbling.”

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