Scientific Meetings

June 26, 2005
Itasca, IL

On Sunday, June 26th, RSRF hosted a Research Update at the Eaglewood Resort and Spa located outside of Chicago. Featured speaker was Dr. Jeffrey Neul, a physician-scientist at the Baylor College of Medicine who is in the unique position of conducting both clinical and basic science research. Dr. Neul sees patients at The Blue Bird Rett Center where he is involved in patient care and clinical trials. He also conducts basic research in the laboratory of one of the world's leading scientists, Dr. Huda Zoghbi.

Dr. Neul began his talk by discussing genetic testing for Rett, a topic of particular interest to parents of children with RTT. In 1999 Huda Zoghbi's lab identified mutations in a gene called MECP2 as the leading cause of Rett Syndrome (RTT). Genes are made up of nucleotide bases, which come in 4 "flavors": A,T,C,G. These long stretches of bases make up the blueprint for proteins. If a DNA sequence contains an error then the protein will also be incorrect. Sequencing means reading the nucleotide bases.

The MECP2 gene can be pictured as a book with 4 chapters. Errors in the gene may include a missing page(s), an extra page(s) or pages in the wrong order. In some cases an entire chapter or two chapters may be missing. This type of mutation is called a deletion. The genetic term for the chapters is exons.

Until March of 2004 it was thought that only mutations in exons 2-3-4 could lead to RTT. In March of 2004 the Bird lab and the Minassian lab announced the discovery of a new form of MeCP2. The new protein, which is 3% longer then the original form, is 10 times more abundant in the brain. This longer section is exon 1. Although mutations in exon 1 are rare testing is now available.

At the Baylor DNA Diagnostic Lab 96% of all patients who are diagnosed clinically with classic RTT have been found to have a mutation or deletion in the MECP2 gene. About 86% of these individuals have small mutations (missing or extra pages) while 10% have deletions (an entire chapter missing). When you include atypical cases the percentage of individuals with mutations or deletions is 91%.

Dr. Neul then went on to discuss whether any correlations exist between symptom severity and specific mutations or deletions. Using the large numbers of RTT individuals that are seen at the Blue Bird Clinic a conclusion was made that the mutation R133C and R134C caused milder symptoms while the R168X caused more serious symptoms. The specific symptoms that were analyzed included head growth, motor function, ambulation, hand use and language. It's important to note that these are general statements since individuals with these specific mutations can present quite differently.

Mutations and deletions in MECP2 can lead to disorders beyond RTT. In fact, they can cause varying degrees of mental retardation with or without seizures, autism, encephalopathy, learning disorders and schizophrenia.

There are 3 scenarios which may lead to males with RTT. One possibility occurs when a boy also has Klinefelter Syndrome (which happens to 1 in 1,000 male births) and therefore has an extra X chromosome (XXY). One of the X's will have the mutation and the other will not. These boys will have symptoms similar to girls with RTT. Another possibility is that a mutation derives not from the sperm or the egg but rather at a slightly later stage, such as the 20-cell stage (blastula). If one cell becomes mutated, every cell downstream to it will also mutate, while the remaining 19 cells and all cells that originate from them will not. This is called somatic mosaicism and the phenotype will resemble a girl with RTT, since the somatic mosaicism will function similarly to X inactivation patterns in girls. The third option occurs when a boy is born with a typical XY karotype and has mutations in MECP2 on all of his X chromosomes. Since he has no healthy copy to mitigate the mutated gene his symptoms will be very severe and likely lead to early death. However, for reasons yet unknown, there are some boys with "milder" mutations.

Dr. Neul continued the research update by discussing the mice models for RTT which include a model that is completely lacking the Mecp2 protein and a model that has a truncated Mecp2 protein. The mice mimic many of the symptoms seen in individuals with RTT including a normal period of development followed by tremors, spasticity, seizures, ataxia, forelimb stereotypies, social/behavior problems, kyphosis/scoliosis.

A discussion of Dr. Rudolf Jaenisch's rescue experiments, funded by RSRF, followed. Dr. Jaenisch devised an experiment to determine the point at which nerve cells become dysfunctional in RTT mice. Early in embryonic development precursor neuronal cells divide rapidly. As the brain cells mature they stop dividing and become post-mitotic. Dr. Jaenisch hooked the Mecp2 gene to the Tau gene which is expressed only in post-mitotic neurons. Mutant Mecp2 mice that also expressed the Tau/Mecp2 transgene never manifested any of the RTT-like symptoms and developed normally. This raises the possibility that neurons are functionally normal in a newborn child and that neural dysfunction manifests itself only later due to prolonged MeCP2 deficiency. If correct, therapeutic strategies aimed at preventing the onset of RTT symptoms could be initiated at birth.

The discussion then turned to a mouse model developed in the Zoghbi lab which overexpresses the human MeCP2 protein. Whereas the knockout mice die by 12 weeks the overexpression model lives past 12 weeks and does not manifest detectable motor/ behavioral abnormalities typically seen in the mutants. Interestingly, by 20-25 weeks these mice show enhanced learning. However between 30 weeks and a year of age the mice begin to deteriorate and die. The take home message is that levels of MeCP2 are tightly regulated and too little or too much lead to severe neurological disease.

MECP2 is believed to function as a transcriptional repressor of its downstream genes. When genes need to be silenced so that a protein is not produced in a given cell, methyl groups attach to CpG dinucleotides, which are often clustered in areas of the gene called CpG islands. MECP2 then binds to these methylated CpG sites, shutting down the gene's production of its protein.

Work by RSRF-funded researcher Janine LaSalle, Ph.D. from UCDavis, suggests that MECP2 comes in two "flavors", high and low expression. Her data suggests that after birth the expression level of MeCP2 becomes higher as the child grows. This helps explain why children with RTT have a normal period of development followed by a regression. RTT symptoms appear only as the child grows and the MeCP2 protein beings to play a crucial role. When the protein is mutated and therefore not functioning properly the symptoms start to appear.

A major breakthrough was made in October of 2003 by RSRF-funded Rudolf Jaenisch of Whitehead Institute and Michael Greenberg of Children's Hospital Boston. The investigators announced the identification of the first downstream gene regulated by MeCP2, called BDNF (brain derived neurotrophic factor). BDNF is an important neuronal growth factor whose expression is regulated in an activity-dependent fashion. Jaenisch and Greenberg discovered that MeCP2 keeps BDNF turned off until stimulated.

A second MeCP2-target gene was announced in December of 2004. With RSRF funding Dr. Terumi Kohwi-Shigematsu of Lawrence Berkley Labs found that MeCP2 regulates a gene called Dlx5. This gene is an imprinting gene which means its expression depends on whether it was maternally or paternally inherited. Mutations in MECP2 "relax" the imprinting of Dlx5, so Dlx5 is expressed more than it should be.

Dlx5 is involved in GABA production which is a major inhibitory neurotransmitter in the brain. If Dlx5 is increased by MeCP2 mutations then expression of GABA might also be increased. This increase in inhibitory neurotransmitter expression might be a contributing factor of RTT symptoms.

Dr. Neul summarized that the MeCP2 protein controls genes in an activity dependent fashion. Furthermore it may control unique sets of genes depending on neuronal populations. It is therefore imperative to devise methodology to identify which genes MeCP2 controls in specific neurons.

With RSRF funding the Zoghbi lab is collaborating with the lab of Nathaniel Heintz at Rockefeller University to study gene expression changes in a mouse model of RTT. Cutting edge technology is being utilized to look at specific neuronal populations suspected of contributing to the symptoms seen in RTT. These populations include the cerebellum (controls movement), hypothalamus (controls homeostasis), dopaminergic neurons (movements), serotonergic neurons (anxiety) and GABAergic neurons (inhibitory). It is likely that this method will elucidate how MECP2 gene mutations lead to RTT symptoms and point to potential therapeutic interventions.

RSRF is grateful to Dr. Neul for participating in our Research Update and for the families and friends who joined us.