Progress Reports for RSRF-Funded Projects Available Online

Interim 1-year progress reports for projects awarded in 2004 are now available online.
Final reports for projects awarded in 2003 will be available in early December.  

Your Participation in the Mutation Databases is Needed

MeCP2.org.uk
The goals of this database are aimed at ascertaining whether correlations exist between specific mutations and certain symptoms and how symptoms change over time. Drs. Brian Hendrich and Skimantas Kriaucionis from the University of Edinburgh invite all parents of children with Rett Syndrome children who have tested positive for MECP2 mutations to submit symptom information directly to their database via an online questionnaire at: www.MeCP2.org.uk. Please note that anyone wishing to submit information will first have to register.

Parents who have previously submitted a questionnaire to the original database, www.homepages.ed.ac.uk/skirmis, will also need to register and submit updated information. All parents are encouraged to submit a questionnaire once a year. This will allow the investigators to keep track of your child's symptoms over time.

InterRett
This project collects information about the features of Rett Syndrome from parents and clinicians. These details are then collated to form an online searchable database. To participate email rett@ichr.uwa.edu.au. You will be sent details about entering your information online.

RettBASE
This database has been constructed by merging mutation and polymorphism data from the published literature pertaining to Rett Syndrome and related clinical disorders, and by incorporating unpublished mutation and polymorphism data that have been submitted directly. Information can only be submitted by clinicians or the testing labs. Please encourage your child's doctor to contribute to this database.

A case of massive gastric necrosis in a young girl with Rett Syndrome

This is the unusual case of a 17-year-old girl with Rett who suffered acute abdominal distension and constipation for a week. Click here to read the abstract.

DNA Binding Selectivity of MeCP2 Due to a Requirement for A/T Sequences Adjacent to Methyl-CpG

This work, by RSRF SAB Chairman Adrian Bird, will aid the discovery of potential MECP2 downstream genes. To read the abstract and lay summary, please click here.

Lost in translation: Translational interference from a recurrent mutation in exon 1 of MECP2

These Australian investigators have made an interesting discovery with regards to the MECP2 isoform discovered last year. To read the abstract and lay summary, please click here.

Splicing mutation associated with Rett syndrome and an experimental approach for genetic diagnosis

A new technique which may help to confirm clinically diagnosed Rett Syndrome patients who, to date, have tested negative for mutations in MECP2. To read the abstract and lay summary, please click here.

Classic Rett syndrome in a boy with R133C mutation of MECP2

Classic Rett syndrome in a boy with R133C mutation of MECP2. To read the abstract and lay summary, please click here.

Survey on ketogenic diet and VNS

RSRF is conducting surveys to gather information on the efficacy of the ketogenic diet and Vagal Nerve Stimualtion (VNS) for children and adults with Rett Syndrome who suffer from intractable seizures. If your child has tried the ketogenic diet, please participate in our survey by clicking here. If your child has a VNS please click here. Data compiled from this survey will be shared with you via the RSRFNewsAlert, website and quarterly newsletter. The data will also be made available to the research community. We thank you in advance for your help.

Children with sudden onset of continuous seizures benefit from propofol treatment

Propofol is a safe and effective drug to treat children with refractory status epilepticus, and it is recommended before thiopental, according to a study in the August 23, 2005 issue of Neurology. Propofol and thiopental are widely used intravenous anesthetics which are also known to help control seizures. Researchers in the Netherlands examined the effects of propofol and thiopental in 33 children (34 episodes total) with refractory status epilepticus. Refractory status epilepticus is a state in which seizures occur in rapid succession without recovery of consciousness between them. These seizures are resistant to treatment and persist for more than 60 minutes. To read this press release please click here. 

The Other Brain Also Deals With Many Woes

Two brains are better than one. At least that is the rationale for the close - sometimes too close - relationship between the human body's two brains, the one at the top of the spinal cord and the hidden but powerful brain in the gut known as the enteric nervous system.

For Dr. Michael D. Gershon, the author of "The Second Brain" and the chairman of the department of anatomy and cell biology at Columbia, the connection between the two can be unpleasantly clear. "Every time I call the National Institutes of Health to check on a grant proposal," Dr. Gershon said, "I become painfully aware of the influence the brain has on the gut."  To read this New York Times article please click here.

Brain Dev. 2005 Sep 14; [Epub ahead of print]
A case of massive gastric necrosis in a young girl with Rett Syndrome
Baldassarre E, Capuano G, Valenti G, Maggi P, Conforti A, Prosperi Porta I.
Pediatric Surgery Unit, Universita di Roma 'La Sapienza', Via del Casalaccio 14, 00046 Grottaferrata Rome, Italy.

Abstract
This is the unusual case of a 17-year-old girl affected by Rett Syndrome (RS) who suffered acute abdominal distension and constipation for a week. Laparotomy showed massive gastric dilatation, with total necrosis and perforation. Total gastrectomy and Y-Roux esophagojejunostomy were performed. We believe the clinical status was caused by the mechanism of air swallowing, present in our patient and typical in RS. In fact, as reported, massive air bloat may result in a decrease of the intramural blood flow with consequential ischemia of the gastric wall. We stress the importance of early detection of the gastroenterological symptoms in these patients, with timely positioning of nasogastric tube and gastrostomy, to prevent serious complications potentially life-threatening as massive gastric necrosis.  

Mol Cell. 2005 Sep 2;19(5):667-78.
DNA Binding Selectivity of MeCP2 Due to a Requirement for A/T Sequences Adjacent to Methyl-CpG
Klose RJ, Sarraf SA, Schmiedeberg L, McDermott SM, Stancheva I, Bird AP.

Abstract
DNA methylation is interpreted by a family of methyl-CpG binding domain (MBD) proteins that repress transcription through recruitment of corepressors that modify chromatin. To compare in vivo binding of MeCP2 and MBD2, we analyzed immunoprecipitated chromatin from primary human cells. Genomic sites occupied by the two MBD proteins were mutually exclusive. As MeCP2 was unable to colonize sites vacated by depletion of MBD2, we tested the hypothesis that methyl-CpG alone is insufficient to direct MeCP2 binding. In vitro selection for MeCP2 bound DNA-enriched fragments containing A/T bases ([A/T](>/=4)) adjacent to methyl-CpG. [A/T](>/=4) was found to be essential for high-affinity binding at selected sites and at known MeCP2 target regions in the Bdnf and Dlx6 genes. MBD2 binding, however, did not require an A/T run. The unexpected restriction of MeCP2 to a defined subset of methyl-CpG sites will facilitate identification of genomic targets that are relevant to Rett Syndrome.

Lay Summary
MeCP2, along with four other proteins (MBD1, MBD2, MBD3, MBD4) are the members of a family of methyl-CpG binding proteins, each of which contains a conserved methyl-DNA binding domain.  Given this similarity, MBD proteins might be expected to compete for methyl-CpG binding sites in the genome.  In an investigation comparing MeCP2 and MBD2 genomic binding, the authors demonstrated that MeCP2 occupies distinct genomic methyl-CpG sites that, for the most part, do not bind MBD2.  Furthermore, MeCP2 is unable to colonize most sites that are vacated by depletion of MBD2.  However, MBD2 is a more promiscuous factor: it can colonize sites vacated by MeCP2.  The difference in binding efficiency is explained by an A/T run of four or more nucleotide bases adjacent to methyl-CpG site required for MeCP2 binding to occur, where MBD2 is indifferent to the presence or absence of an A/T run.  Further mutation analyses confirmed that the AT sequence adjacent to a methyl-CpG is necessary for MeCP2 binding.  In addition, it was demonstrated that MeCP2 binds to the DLX6 and Bdnf target genes at such sequences.  The surprising nature of MeCP2 binding sites will facilitate identification of genomic targets that may be relevant to Rett syndrome. To read the RSRF press release regarding this discovery please click here.  

J Med Genet. 2005 Sep 9; [Epub ahead of print]
Lost in translation: Translational interference from a recurrent mutation in exon 1 of MECP2
Saxena A, de Lagarde D, Leonard H, Williamson S, Vasudevan V, Christodoulou J, Thompson E, Macleod P, Ravine D.
University of Western Australia, Australia

Abstract
Rett syndrome (RTT) is an X- linked neuro-developmental disorder affecting mostly girls. Mutations in the coding region of MECP2 are found in 80% of classic RTT patients. Until recently, the region encoding MECP2 was believed to comprise exons 2, 3 and 4 with the ATG start site located at the end of exon 2 (MeCP2_e2). Another mRNA transcript transcribed from exon 1 (MeCP2_e1) has been recently reported. METHODS: We screened exon 1 among RNA samples from 20 females with classic or atypical RTT. RESULTS: A previously reported 11 base deletion in exon 1 was detected in one subject with a milder phenotype. Although RNA expression for both protein isoforms was detected from the mutant allele, evaluation of MeCP2 protein in uncultured patient lymphocytes by immunocytochemistry revealed that MeCP2 protein production was restricted to only 74-76 % of lymphocytes. X chromosome inactivation studies of genomic DNA revealed similar XCI ratios at the HUMARA locus (73:27 with HpaII and 74:26 with McrBc). DISCUSSION: We have demonstrated that translation but not transcription of the MeCP2_e2 isoform is ablated by the 11 nucleotide deletion, 103 nucleotides upstream of the MeCP2_e2 translation start site. These findings reveal that nucleotides within the deleted sequence in the 5'-UTR of the MeCP2_e2 transcript, while not required for transcription, are essential for translation.

Lay Summary
Cells use the two-step process of transcription and translation to read each gene and produce the string of amino acids that makes up a protein. The authors investigated MeCP2 levels at both the transcription and translation phase in an individual with a mild Rett phenotype.  The patient has an 11 base deletion in exon 1 generating a severely truncated MeCP2 protein. You may remember that in April of 2004 two independent labs discovered an alternate form of the MeCP2 protein which is 19 amino acids longer (which comprises exon 1). The new isoform, called MeCP2e1, is found more abundantly in brain then the original isoform, called MeCP2e2. Interestingly, the investigators found that despite the exon 1 mutation RNA expression from both isoforms were detected (the translation phase). However, the mutated protein was not found (the translation phase). These findings show that the deleted nucleotides in exon 1 are not crucial for transcriptions but are essential for translation. In other words. exon 1 plays a role in translating the MeCP2 protein. A helpful interactive diagram can be found at this site for readers interested in learning more about transcription and translation.  

Hum Genet. 2005 Aug 17;:1-8 [Epub ahead of print]
Splicing mutation associated with Rett syndrome and an experimental approach for genetic diagnosis
Abuhatzira L, Makedonski K, Galil YP, Gak E, Zeev BB, Razin A, Shemer R.
Department of Cellular Biochemistry and Human Genetics, Hadassah Medical School, The Hebrew University, Jerusalem, Israel, 91120, shemer@md2.huji.ac.il.

Abstract
Around 80% of Rett syndrome (RS) cases have a mutation or deletion within the coding sequence of the MeCP2 gene. The other RS patients remain genetically undiagnosed. A significant fraction (10-15%) of disease-causing mutations in humans, affect pre-mRNA splicing. Two potential splice mutations were found in the MeCP2 gene in RS patients, however it was not clear whether these mutations in fact interfere with splicing and consequently cause RS. One such mutation is a deletion of the GT dinucleotide at the 5' donor splice site of exon 1 and the other a deletion of the T nucleotide in the polypyrimidine tract (PPT) of intron 3. Here we experimentally assess the effects exerted by these mutations on the expression of MeCP2 in patients' blood samples and on splicing of the MeCP2 transcript using a hybrid minigene in transient transfection experiments. The results revealed that the Delta T mutation in the PPT is a benign polymorphism and that the GT deletion in intron 1 is a bona fide splicing mutation that causes a complete skipping of exon 1 in the minigene transfection experiment. This explains the observed complete elimination of the MeCP2 message and protein in the lymphoblast clones of the RS patient that carry the mutation on the active X. An analysis of the MeCP2 transcript and protein production in lymphoblast clones, as described here, can be used to confirm clinically diagnosed RS patients with no mutation in the MeCP2 coding sequence. This will enable RS diagnosis without specifically identifying a mutation.

Lay Summary
Genes are made up of exons and introns. When proteins are made the introns are spliced out. Exons can also be spliced out resulting in various isoforms of the same protein. Abnormalities in splicing account for 10-15% of all gene mutations that cause diseases.  In order to distinguish between a harmless polymorphism (a change in the sequence of DNA associated with a large portion of the population) and a pathogenic splice mutation, expression analyses at the RNA and protein level are often required.   By looking at the expression of MeCP2 gene and protein in patient blood samples, and by recreating the mutations in a cell culture system, the experiments described in this paper test whether two identified MeCP2 gene mutations located at sites important for its proper splicing, are in fact responsible for the production of mutant MeCP2, or are benign.  The first mutation is a deletion of the first two nucleotides of intron 1, thereby eliminating a recognition site for the RNA splicing machinery.  The second mutation is a deletion in a region of intron 3; a site similarly essential for MeCP2 splicing.  The results demonstrate that the former mutation is indeed causative, resulting in aberrant MeCP2 splicing, whereas the latter mutation is a benign polymorphism. This technique could help to confirm clinically diagnosed Rett Syndrome patients who, to date, have tested negative for mutations in MECP2.  

Brain Dev. 2005 Sep;27(6):439-42.
Classic Rett syndrome in a boy with R133C mutation of MECP2
Masuyama T, Matsuo M, Jing JJ, Tabara Y, Kitsuki K, Yamagata H, Kan Y, Miki T, Ishii K, Kondo I.
Department of Pediatrics, Faculty of Medicine, Saga University, Saga, Japan.

Abstract
About 80% of female patients with Rett syndrome (RTT) display a mutation in the methyl-CpG-binding protein 2 (MECP2) gene, but most males with MECP2 mutation experience severe fatal encephalopathy or non-specific X-linked mental retardation (XLMR). The existence of male RTT has been extensively discussed. We report herein a boy with classic RTT in a family with a missense mutation in MECP2. The mother exhibited slight mental retardation and was a carrier for R133C. The patient could stand with support at 12-months-old, and stereotypic hand movements appeared at 3-years-old. He became bed-ridden by 8-years-old. The R133C mutation was present in MECP2 without somatic mosaicism. A sister with R133C displayed classic RTT. The R133C mutation has been detected in female patients with classic and preserved speech variant RTT, but not in males with non-specific XLMR. These results suggest that clinical phenotypes caused by DNA mutation in MECP2 are determined by position of the mutation in the gene, and R133 represents a critical amino acid residue in the induction of RTT symptoms in humans.

Lay Summary
The existence of Rett syndrome in boys has been extensively discussed.  Most males with the MeCP2 mutation experience severe fatal encephalopathy or non-specific X-linked mental retardation.  In this case report, the authors document a boy with classic Rett syndrome in a family with a missense mutation in MeCP2, R133C.  The patient could stand with support at 12 months, developed stereotypic hand movements at 3-years-old, and was bed-ridden by 8-years-old.  The boy has a normal 46, XY karyotype and a lack of wild-type MeCP2 expression, eliminating the possibility of somatic mosaicism or an XXY karyotype.  Interestingly, the mother exhibits slight mental retardation and is a carrier for R133C, and a sister with R113C displays classic Rett syndrome.  

The Other Brain Also Deals With Many Woes
By HARRIET BROWN
August 23, 2005 New York Times

Two brains are better than one. At least that is the rationale for the close - sometimes too close - relationship between the human body's two brains, the one at the top of the spinal cord and the hidden but powerful brain in the gut known as the enteric nervous system.

For Dr. Michael D. Gershon, the author of "The Second Brain" and the chairman of the department of anatomy and cell biology at Columbia, the connection between the two can be unpleasantly clear. "Every time I call the National Institutes of Health to check on a grant proposal," Dr. Gershon said, "I become painfully aware of the influence the brain has on the gut."

In fact, anyone who has ever felt butterflies in the stomach before giving a speech, a gut feeling that flies in the face of fact or a bout of intestinal urgency the night before an examination has experienced the actions of the dual nervous systems.

The connection between the brains lies at the heart of many woes, physical and psychiatric. Ailments like anxiety, depression, irritable bowel syndrome, ulcers and Parkinson's disease manifest symptoms at the brain and the gut level.

"The majority of patients with anxiety and depression will also have alterations of their GI function," said Dr. Emeran Mayer, professor of medicine, physiology and psychiatry at the University of California, Los Angeles.

A study in 1902 showed changes in the movement of food through the gastrointestinal tract in cats confronted by growling dogs.

One system's symptoms - and cures - may affect the other. Antidepressants, for example, cause gastric distress in up to a quarter of the people who take them. Butterflies in the stomach are caused by a surge of stress hormones released by the body in a "fight or flight" situation. Stress can also overstimulate nerves in the esophagus, causing a feeling of choking.

Dr. Gershon, who coined the term "second brain" in 1996, is one of a number of researchers who are studying brain-gut connections in the relatively new field of neurogastroenterology. New understandings of the way the second brain works, and the interactions between the two, are helping to treat disorders like constipation, ulcers and Hirschprung's disease.

The role of the enteric nervous system is to manage every aspect of digestion, from the esophagus to the stomach, small intestine and colon. The second brain, or little brain, accomplishes all that with the same tools as the big brain, a sophisticated nearly self-contained network of neural circuitry, neurotransmitters and proteins.

The independence is a function of the enteric nervous system's complexity.

"Rather than Mother Nature's trying to pack 100 million neurons someplace in the brain or spinal cord and then sending long connections to the GI tract, the circuitry is right next to the systems that require control," said Jackie D. Wood, professor of physiology, cell biology and internal medicine at Ohio State.

Two brains may seem like the stuff of science fiction, but they make literal and evolutionary sense.

"What brains do is control behavior," Dr. Wood said. "The brain in your gut has stored within its neural networks a variety of behavioral programs, like a library. The digestive state determines which program your gut calls up from its library and runs."

When someone skips lunch, the gut is more or less silent. Eat a pastrami sandwich, and contractions all along the small intestines mix the food with enzymes and move it toward the lining for absorption to begin. If the pastrami is rotten, reverse contractions will force it - and everything else in the gut - into the stomach and back out through the esophagus at high speed.

In each situation, the gut must assess conditions, decide on a course of action and initiate a reflex.

"The gut monitors pressure," Dr. Gershon said. "It monitors the progress of digestion. It detects nutrients, and it measures acid and salts. It's a little chemical lab."

The enteric system does all this on its own, with little help from the central nervous system.

The enteric nervous system was first described in 1921 by Dr. J. N. Langley, a British physician who believed that it was one of three parts - along with the parasympathetic and sympathetic nervous systems - of the autonomic nervous system, which controls involuntary behaviors like breathing and circulation. In this triad, the enteric nervous system was seen as something of a tag-along to the other two.

After Langley died, scientists more or less forgot about the enteric nervous system. Years later, when Dr. Gershon reintroduced the concept and suggested that the gut might use some of the same neurotransmitters as the brain, his theory was widely ridiculed.

"It was like saying that New York taxi drivers never miss a showing of 'Tosca' at the Met," he recalled.

By the early 80's, scientists had accepted the idea of the enteric nervous system and the role of neurotransmitters like serotonin in the gut.

It is no surprise that there is a direct relationship between emotional stress and physical distress. "Clinicians are finally acknowledging that a lot of dysfunction in GI disorders involves changes in the central nervous system," said Gary M. Mawe, a professor of anatomy and neurobiology at the University of Vermont.

The big question is which comes first, physiology or psychology?

The enteric and central nervous systems use the same hardware, as it were, to run two very different programs. Serotonin, for instance, is crucial to feelings of well-being. Hence the success of the antidepressants known as S.S.R.I.'s that raise the level of serotonin available to the brain.

But 95 percent of the body's serotonin is housed in the gut, where it acts as a neurotransmitter and a signaling mechanism. The digestive process begins when a specialized cell, an enterochromaffin, squirts serotonin into the wall of the gut, which has at least seven types of serotonin receptors. The receptors, in turn, communicate with nerve cells to start digestive enzymes flowing or to start things moving through the intestines.

Serotonin also acts as a go-between, keeping the brain in the skull up to date with what is happening in the brain below. Such communication is mostly one way, with 90 percent traveling from the gut to the head.

Many of those messages are unpleasant, and serotonin is involved in sending them. Chemotherapy drugs like doxorubicin, which is used to treat breast cancer, cause serotonin to be released in the gut, leading to nausea and vomiting. "The gut is not an organ from which you wish to receive frequent progress reports," Dr. Gershon said.

Serotonin is also implicated in one of the most debilitating gut disorders, irritable bowel syndrome, or I.B.S., which causes abdominal pain and cramping, bloating and, in some patients, alternating diarrhea and constipation.

"You can run any test you want on people with I.B.S., and their GI tracts look essentially normal," Dr. Mawe said. The default assumption has been that the syndrome is a psychosomatic disease.

But it turns out that irritable bowel syndrome, like depression, is at least in part a function of changes in the serotonin system. In this case, it is too much serotonin rather than too little.

In a healthy person, after serotonin is released into the gut and initiates an intestinal reflex, it is whisked out of the bowel by a molecule known as the serotonin transporter, or SERT, found in the cells that line the gut wall.

People with irritable bowel syndrome do not have enough SERT, so they wind up with too much serotonin floating around, causing diarrhea.

The excess serotonin then overwhelms the receptors in the gut, shutting them down and causing constipation.

When Dr. Gershon, whose work has been supported by Novartis, studied mice without SERT, he found that they developed a condition very much like I.B.S. in humans.

Several new serotonin-based drugs - intestinal antidepressants, in a way - have brought hope for those with chronic gut disorders.

Another mechanism that lends credence to physiology as the source of intestinal dysfunctions is the system of mast cells in the gut that have an important role in immune response.

"During stress, trauma or 'fight or flight' reactions, the barrier between the lumen, the interior of the gut where food is digested, and the rest of the bowel could be broken, and bad stuff could get across," Dr. Wood said. "So the big brain calls in more immune surveillance at the gut wall by activating mast cells."

These mast cells release histamines and other inflammatory agents, mobilizing the enteric nervous system to expel the perceived intruders, and causing diarrhea.

Inflammation induced by mast cells may turn out to be crucial in understanding and treating GI disorders. Inflamed tissue becomes tender. A gut under stress, with chronic mast cell production and consequent inflammation, may become tender, as well.

In animals, Dr. Mawe said, inflammation makes the sensory neurons in the gut fire more often, causing a kind of sensory hyperactivity. "I have a theory that some chronic disorders may be caused by something like attention deficit disorder in the gut," he said.

Dr. Gershon, too, theorizes that physiology is the original culprit in brain-gut dysfunctions. "We have identified molecular defects in the gut of everyone who has irritable bowel syndrome," he said. "If you were chained by bloody diarrhea to a toilet seat, you, too, might be depressed."

Still, psychology clearly plays a role. Recent studies suggest that stress, especially early in life, can cause chronic GI diseases, at least in animals. "If you put a rat on top of a little platform surrounded by water, which is very stressful for a rat, it develops the equivalent of diarrhea," Dr. Mayer said.

Another experiment showed that when young rats were separated from their mothers, the layer of cells that line the gut, the same barrier that is strengthened by mast cells during stress, weakened and became more permeable, allowing bacteria from the intestine to pass through the bowel walls and stimulate immune cells.

"In rats, it's an adaptive response," Dr. Mayer said. "If they're born into a stressful, hostile environment, nature programs them to be more vigilant and stress responsive in their future life."

He said up to 70 percent of the patients he treats for chronic gut disorders had experienced early childhood traumas like parents' divorces, chronic illnesses or parents' deaths. "I think that what happens in early life, along with an individual's genetic background, programs how a person will respond to stress for the rest of his or her life," he said.

Either way, what is good for one brain is often good for the other, too. A team of researchers from Penn State University recently discovered a possible new direction in treating intestinal disorders, biofeedback for the brain in the gut.

In an experiment published in a recent issue of Neurogastroenterology and Motility, Robert M. Stern, a professor of psychology at Penn State, found that biofeedback helped people consciously increase and enhance their gastrointestinal activity. They used the brains in their heads, in other words, to help the brains in their guts, proving that at least some of the time two brains really are better than one.