Saturday, November 21, 2009

Pictures of Indian Rett syndrome Awareness meet

We organised Rett syndrome Awareness Meet on 11th October, 2009 in our Dept. of Pediatrics, All India Institute of Medical sciences, New Delhi, to raise more Awareness.

This event was attended by around 40 Indian families from different parts of India (Majority from New Delhi)and Doctors, Scientists, Researchers and other healthcare professionals. We were happy to see the response shown by the families in this regard and we are really thankful to them for helping us in this cause and making this event successful with their presence.

This Awareness meet was organised for the second time as the first awareness meet was organised on 19th October, 2008 and that was also a great ans successful event.

We wish to keep on doing these event to raise more awareness in India as still people are not knowing much about this disorder. We wish that one day these angel be recognised by all and a treatment will come out which will help the Angels to lead a quality life.

Prayers for Care,

Rajni Khajuria

Rett syndrome Awareness(India)(

FAQ on Gene Therapy turnaround (Posted by Victoria Stern)

Judging by the stream of studies in the last few months, it seems the field of gene therapy is beginning to replace its troubled history with the beginnings of a promising future.

In September, researchers reported that viral delivery of a pigment gene allowed colorblind squirrel monkeys to see red and green for the first time, providing hopes that the technique could be used to treat colorblindness in humans. In October, transplant scientists showed they could bolster the health of donor lungs by supplying a gene coding for an anti-inflammatory molecule. Earlier this month came a report in which gene therapy was used to halt the progression of adrenoleukodystrophy, a fatal brain disease, in two young boys using a virus derived from HIV to deliver the gene for the missing enzyme. Finally, a study published today (November 11) in Science Translational Medicine reports that administering a gene blocking muscle breakdown to monkeys boosts the size and strength of their muscles -- suggesting such a treatment may one day help patients with degenerative muscle disorders.

With the flurry of recent successes, Mark Kay, director of the Human Gene Therapy program at Stanford University School of Medicine and one of the founders of the American Society of Gene and Cell Therapy, believes that "the mood in the field is pretty positive." Kay took time to chat with The Scientist about the progress that's been made in the field the hurdles still left to overcome.

The Scientist: Can you start by describing the outlook for gene therapy after these recent successes?

Mark Kay: These studies have provided a great model for the potential success of gene therapeutics. There is a lot of optimism in the gene therapy field right now that these therapies will work well. In the course of gene therapy research, there have been a lot of unanticipated hurdles, but in a relatively short time [since trials began in the 1980s], I think we have made a lot of progress.

TS: What kinds of conditions are best suited for gene therapy?

MK: There are a lot of conditions for which gene therapy could work. The easiest target appears to be single gene disorders, including adrenoleukodystrophy and blindness, where only a small percentage of cells need to be fixed.

Other disorders caused by a single gene are more complicated to treat, however. For instance, Duchene muscular dystrophy, which affects all the skeletal and cardiac muscle, requires correcting one gene, but the defect needs to be changed in a large number of cells to make a clinical treatment successful. Cancers, which deal with many different cells in the body, will also be difficult to cure with gene therapy, but perhaps when used in combination with other treatments, gene therapy may turn out to have therapeutic results.

TS: What do you see as the main technical hurdles that gene therapeutics has yet to overcome?

MK: I would lay out four main hurdles that we have to overcome:

1) First, it's essential to get a vector to the specific cells in high enough levels to produce the desired response but not have toxicity problems.

2) Once the vector with the encoded gene reaches the desired cell, it must be internalized and get to the nucleus. The cell has naturally implemented a lot of barriers to prevent this new DNA from interacting with the existing DNA, but viruses are good delivery tools because they have developed mechanisms to get passed these hurdles. Overcoming this difficulty has turned out to be more difficult than previously anticipated.

3) Once in nucleus, the new gene must be able to persist for the desired amount of time. The cell can sometimes shut the new gene off, making it ineffective.

4) The final main problem is the potential for an immune response. If the gene product is recognized as a foreign agent, the body can mount an attack against the vector or the protein.

Another general issue is determining how long the gene therapy should persist. If you're treating an infection or cancer, you only need gene therapy to last until the infection or cancer has been eliminated. But in genetic disorders, you need lifelong gene correction.

TS: Are there still safety concerns?

MK: People are always going to be cautious moving forward. We don't know for sure what the risks of some of these treatments might be. For instance, there is increasing evidence that the patients who developed leukemia [two children whose X-linked Severe Combined Immunodeficiency Disease was cured with gene therapy in the highly publicized 1998 trial later developed leukemia] may have a very disease-specific type of problem. Any time you're inserting new DNA into cells there will always be some risk that it will increase the chances of developing a malignancy. There are many medical treatments unrelated to gene therapy where the risk of developing cancer is reasonably high. For example, by treating one type of cancer, you may be using molecules that increase your risk of developing another type of cancer. As we continue to move into unknown territory in gene therapy, it will be important to watch for toxicity and longer-term side effects.

TS: What ethical issues remain that the field will still have to address?

MK: The bigger ethical dilemmas will likely come in the future when we have to decide where to draw the line in terms of what conditions we're willing to treat. Clearly gene therapy should be used to combat severe mental disorders or genetic diseases, but deciding whether to treat neurobehavioral disorders, such as depression or addiction, provides more of a gray area. And should we use gene therapy to enhance or select for certain traits, like higher IQ or athletic ability. Even though it's not possible to treat any of these disorders now, with more advances in gene therapy, people may start asking these questions in the future.

TS: What's the next frontier in the gene therapy?

MK: While most of gene therapy is focused on adding another copy of a functional gene, another path may be to design molecules that can turn off defective genes. For instance, in Huntington's disease, gene therapy could be used to turn off the defective gene that produces an abnormal protein.

I think one of the most important aspects for a lot of these diseases is to treat them early on, before the pathology has caused irreversible damage. Prevention is much more effective because it's much harder to fix a problem in a neurodegenerative disease or muscular dystrophy when the damage has already been done.

Editor's note (November 12): This post originally referred to the American Society of Gene Therapy. That organization changed its name to the American Society of Gene and Cell Therapy in May, 2009.The post has been updated with the current name.

Source: The Magazine

Gene therapy technique slows brain disease

A strategy that combines gene therapy with blood stem cell therapy may be a useful tool for treating a fatal brain disease, French researchers have found. These findings appear in the 6 November 2009 issue of the journal Science, which is published by AAAS, the nonprofit science society.

In a pilot study of two patients monitored for two years, an international team of researchers slowed the onset of the debilitating brain disease X-linked adrenoleukodystrophy (ALD) using a lentiviral vector to introduce a therapeutic gene into patient's blood cells. Although studies with larger cohorts of patients are needed, these results suggest that gene therapy with lentiviral vectors, which are derived from disabled versions of human immunodeficiency virus (HIV), could potentially become instrumental in treating a broad range of human disorders.

"This is the first time we were able to successfully use an HIV-derived lentivirus vector for gene therapy in humans, and also the first time that a very severe brain disease has been treated with efficacy by gene therapy. We've demonstrated that this HIV-derived lentivirus vector works as was hoped for so many years," said coauthor Patrick Aubourg, professor of pediatrics at University Paris-Descartes and head of a research unit at Inserm-University Paris Descartes.

IMAGE: This figure represents four cells, four purified CD34+ (the CD34+ cell population comprises true hematopoetic stem cell) from patient P1. These cells were sampled/purifed from his bone marrow 2 years after gene...

Click here for more information.

Featured in the movie "Lorenzo's Oil," ALD is a severe hereditary condition caused by a deficiency of a protein called ALD that is involved in fatty acid degradation. Sufferers steadily lose their myelin sheath, the protective layer that coats nerve fibers in the brain. Without myelin the nerves lose function, leading to increasing physical and mental disability in patients. X-linked ALD, the most common form of the disease, affects boys starting at age 6-8 years of age and death usually occurs before the patients reach adolescence.

Bone marrow transplants typically slow progression of the disease because the donor marrow includes cells that develop into myelin-producing cells. However, finding a matching bone marrow donor can be a challenging and lengthy process, and the procedure carries considerable risks.

Genetically correcting the blood stem cells in the patients' own bone marrow may prove to be a valuable alternative approach when no matched donors are available.

In most gene therapy studies, a working gene is inserted into the genome to replace a dysfunctional, disease-causing gene. A carrier molecule called a vector is used to deliver the therapeutic gene into the patient's cells. Vectors are typically the backbones of viruses that have been genetically altered to carry normal human DNA. Scientists have recently turned to vectors based on the lentivirus genus of retroviruses, which includes HIV. Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells, and are thought to provide long-term and stable gene expression, unlike other retroviruses.

"The HIV-derived lentivirus vector allows expression of the therapeutic gene in principle for life, because the therapeutic gene is inserted in the chromosomes—the genome. Therefore, cells that derive from the initially corrected cells, stem cells in particular, will continue to express the therapeutic gene forever," said Aubourg.

IMAGE: Progeny of HSCs that were engineered to carry the correct version of a gene (through the integration of a lentiviral vector) distribute throughout the body. Cartier et al. show that some cells...

Click here for more information.

In the study, blood stem cells were removed from the patients and genetically corrected in the lab, using a lentiviral vector to introduce a working copy of the ALD gene into the cells. The modified cells were then infused back into the patients' after they had received a treatment that destroyed their bone marrow. Two years later, healthy ALD proteins were still detectable in both patients' blood cells. Encouragingly, both patients showed neurological improvement and a delay in disease progression comparable to that seen with bone marrow transplants.

The healthy ALD protein was expressed in about 15 percent of blood cells, yet surprisingly this low level was sufficient to slow brain disease in ALD. "This percentage of correction will not be sufficient for all diseases," warns Aubourg. "There is a lot of work to be done to make this gene therapy vector more powerful, less complicated, and less expensive. This is only the beginning," he said.

Gene therapy is not without serious risks. Like other retrovirus vectors, the HIV-derived lentivirus vector is tasked with inserting the therapeutic gene in the chromosomes of the patients' cells. In a worst case scenario, this action could disturb the biology of the cells and patients could end up with leukemia; this outcome has occurred in past gene therapy trials. "The HIV-derived lentivirus vector basically has this same risk, although the design of the vector makes patients less prone to this side effect," said Aubourg.


This research was funded by INSERM (National Institute of Health and Research Medical), Assistance Publique des Hôpitaux de Paris, PHRC programs, the Deutsche Forchungsgemeinschaft and the German Ministry of Education and Research, the European Leukodystrophy Association, the Association Française contre les Myopathies, the Stop ALD Foundation and University Paris-Descartes.

Related Press Briefing in France: A press conference related to the forthcoming Science paper, "Hematopoietic Stem Cell Gene Therapy with a Lentiviral Vector in X-linked Adrenoleukodystrophy," by Dr. Nathalie Cartier and colleagues is planned for 2:00 p.m. in Paris on Wednesday, 4 November.

All information released during the press conference will remain under embargo until 2:00 p.m. U.S. ET/8:00 p.m. CET Thursday, 5 November 2009.

Speakers include Drs. Nathalie Cartier and Patrick Aubourg from l'Institut National de la Santé et de la recherche médicale (INSERM) and the Hopital Saint-Vincent de Paul in Paris, France, Dr. Christof von Kalle from the National Center for Tumor Diseases and German Cancer Research Center in Heidelberg, Germany, and Dr. Salima Hacein-Bey-Abina from Hôpital Necker Enfants-Malades in Paris, France who will present at INSERM, Meeting Room 132 (10th floor) 101 rue de Tolbiac, Paris 13ème (Web site:; Metro: Olympiades Line 14 or Metro Tolbiac Line 7). For further directions, please email

The embargoed press briefing, for journalists only, is being organized by the journal Science and its publisher, the nonprofit American Association for the Advancement of Science (AAAS), in cooperation with INSERM.

Reporters interested in attending the briefing are asked to send an e-mail to requesting pre-registration, or to request materials, please contact Natasha Pinol, senior communications officer, at email

An audio file will be available on the Science Press Package website at (please enter your username and passcode).

The American Association for the Advancement of Science (AAAS) is the world's largest general scientific society and publisher of the journal, Science (, Science Signaling (, and Science Translational Medicine ( AAAS was founded in 1848, and serves 262 affiliated societies and academies of science, reaching 10 million individuals. Science has the largest paid circulation of any peer-reviewed general science journal in the world, with an estimated total readership of 1 million. The non-profit AAAS ( is open to all and fulfills its mission to "advance science and serve society" through initiatives in science policy; international programs; science education; and more. For the latest research news, log onto EurekAlert!,, the premier science-news Web site, a service of AAAS.

Contact: Natasha Pinol
American Association for the Advancement of Science

Is scoliosis surgery in your child's future? (By Etan Sugarman and Vishal Sarwahi, MD)

Acute Pancreatitis after Scoliosis Surgery
By Etan Sugarman and Vishal Sarwahi, MD
Spine Deformity Surgery Service
Department of Orthopaedic Surgery
Children's Hospital at Montefiore and the Albert Einstein College of Medicine

It is estimated that 14% - 30% of patients undergoing surgery for scoliosis develop acute pancreatitis in the immediate post-operative period. While a number of papers have been published, the cause is poorly understood and risk factors are not well established. Intra-operative blood loss, low body-mass index (BMI), calcium chloride administration, gastro-esophageal reflux (GERD) and reactive airway disease have been implicated. The incidence is largely felt to be similar in children with idiopathic and neuromuscular scoliosis.

Acute pancreatitis is defined as an acute inflammation of the pancreas. As the process develops pancreatic enzymes (amylase and lipase), normally secreted into the intestine to digest food, spill onto the pancreas itself and produce severe inflammation. Levels of these enzymes increase dramatically, and elevations greater than three times normal are considered diagnostic for acute pancreatitis. However, enzymes may also be elevated without any signs or symptoms, or may not be sufficiently elevated to make the diagnosis. In these instances an ultrasound may establish the presence or absence of acute pancreatitis.

Clinically acute pancreatitis presents as severe upper abdominal pain, tenderness, nausea, and vomiting. Treatment involves fluid hydration, resting the bowel, and adequate pain management. Data suggests that less than 1% of children will have long term complications. These include recurrence, chronic pancreatitis, and pancreatic pseudocysts and need follow up.

Acute pancreatitis has been well documented as a complication not only in spinal fusion surgery, but also in most abdominal surgeries, as well as cardiac and transplant surgeries. Awareness is the key. In our experience at the Children's Hospital at Montefiore, we have a lower than reported rate of post-operative pancreatitis. To minimize risk factors we employ a team concept early on in patient care, nutritional and GI consultation, blood conservation techniques during surgery and maintain heightened awareness especially in children with special needs. This coupled with a periodic clinical and laboratory evaluation help us detect acute pancreatitis early on in its course and helps decrease hospital stay and its impact on our patients and their families.

Source: Rett syndrome research trust

We are all mutants

Measurement of mutation rate in humans by direct sequencing
This release is available in Chinese.

An international team of 16 scientists today reports the first direct measurement of the general rate of genetic mutation at individual DNA letters in humans. The team sequenced the same piece of DNA - 10,000,000 or so letters or 'nucleotides' from the Y chromosome - from two men separated by 13 generations, and counted the number of differences. Among all these nucleotides, they found only four mutations.

In 1935 one of the founders of modern genetics, J. B. S. Haldane, studied men in London with the blood disease haemophilia and estimated that there would be one in 50,000 incidence of mutations causing haemophilia in the gene affected - the equivalent of a mutation rate of perhaps one in 25 million nucleotides across the genome. Others have measured rates at a few further specific genes or compared DNA from humans and chimpanzees to produce general estimates of the mutation rate expressed more directly in nucleotides of DNA.

Remarkably, the new research, published today in Current Biology, shows that these early estimates were spot on - in total, we all carry 100-200 new mutations in our DNA. This is equivalent to one mutation in each 15 to 30 million nucleotides. Fortunately, most of these are harmless and have no apparent effect on our health or appearance.

"The amount of data we generated would have been unimaginable just a few years ago," says Dr Yali Xue from the Wellcome Trust Sanger Institute and one of the project's leaders. "But finding this tiny number of mutations was more difficult than finding an ant's egg in the emperor's rice store."

Team member Qiuju Wang recruited a family from China who had lived in the same village for centuries. The team studied two distant male-line relatives - separated by thirteen generations - whose common ancestor lived two hundred years ago.

To establish the rate of mutation, the team examined an area of the Y chromosome. The Y chromosome is unique in that, apart from rare mutations, it is passed unchanged from father to son; so mutations accumulate slowly over the generations.

Despite many generations of separation, researchers found only 12 differences among all the DNA letters examined. The two Y chromosomes were still identical at 10,149,073 of the 10,149,085 letters examined. Of the 12 differences, eight had arisen in the cell lines used for the work. Only four were true mutations that had occurred naturally through the generations.

We have known for a long time that mutations occur occasionally in each of us, but have had to guess exactly how often. Now, thanks to advances in the technology for reading DNA, this new research has been possible.

Understanding mutation rates is key to many aspects of human evolution and medical research: mutation is the ultimate source of all our genetic variation and provides a molecular clock for measuring evolutionary timescales. Mutations can also lead directly to diseases like cancer. With better measurements of mutation rates, we could improve the calibration of the evolutionary clock, or test ways to reduce mutations, for example.

Even with the latest DNA sequencing technology, the researchers had to design a special strategy to search for the vanishingly rare mutations. They used next-generation sequencing to establish the order of letters on the two Y chromosomes and then compared these to the Y chromosome reference sequence.

Having identified 23 candidate SNPs - or single letter changes in the DNA - they amplified the regions containing these candidates and checked the sequences using the standard Sanger method. A total of four naturally occurring mutations were confirmed. Knowing this number of mutations, the length of the area that they had searched and the number of generations separating the individuals, the team were able to calculate the rate of mutation.

"These four mutations gave us the exact mutation rate - one in 30 million nucleotides each generation - that we had expected," says the study's coordinator, Chris Tyler-Smith, also from The Wellcome Trust Sanger Institute. "This was reassuring because the methods we used - harnessing next-generation sequencing technology - had not previously been tested for this kind of research. New mutations are responsible for an array of genetic diseases. The ability to reliably measure rates of DNA mutation means we can begin to ask how mutation rates vary between different regions of the genome and perhaps also between different individuals."


Notes to Editors

Publication Details

Xue Y et al. (2009) Human Y chromosome base substitution mutation rate measured by direct sequencing in a deep-rooting pedigree. Current Biology.

This work was supported by the Joint Project from the NSFC and The Royal Society, and the Wellcome Trust.

Participating Centres

The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK
Department of Otorhinolaryngology-Head and Neck Surgery, and Institute of Otolaryngology, Chinese People's Liberation Army General Hospital, Beijing, China
Beijing Genomics Institute at Shenzhen, Shenzhen, China

The Wellcome Trust Sanger Institute, which receives the majority of its funding from the Wellcome Trust, was founded in 1992. The Institute is responsible for the completion of the sequence of approximately one-third of the human genome as well as genomes of model organisms and more than 90 pathogen genomes. In October 2006, new funding was awarded by the Wellcome Trust to exploit the wealth of genome data now available to answer important questions about health and disease.

The Wellcome Trust is the largest charity in the UK. It funds innovative biomedical research, in the UK and internationally, spending over £600 million each year to support the brightest scientists with the best ideas. The Wellcome Trust supports public debate about biomedical research and its impact on health and wellbeing.


Tuesday, November 17, 2009

Rett syndrome and Epilepsy

Rett syndrome
This syndrome happens in about one in every 10,000-12,000 girls. It may rarely affect boys. Boys are always affected far more severely than girls. The cause is due to a genetic abnormality, called a ‘mutation’. This abnormality or mutation is usually found on one of the sex chromosomes – the X chromosome. The most common mutation is called the MECP2 mutation. A different and much less commonly found genetic mutation is nearly always seen in those children with Rett syndrome who have a very severe type of epilepsy.

Girls with Rett Syndrome may show normal development for the first six or more months of their life. Then between six months and 30 months of age (often between six-18 months), their development slows down and may even go backwards. They become less interested in play, lose the ability to speak and may become irritable and scream for no obvious reason. They stop using their hands purposefully and they may, instead, begin to move their hands in a repetitive way, often with hand-wringing or hand-washing movements.

Epilepsy eventually happens in about 70 per cent of those children. It usually begins after the age of two years but may begin in the first year of life.

The seizures may be of various types including generalised convulsive (tonic-clonic) seizures, absences (where the child goes blank), myoclonic (jerk) seizures and tonic seizures (where the child stiffens).

A particular type of seizure called infantile spasms may also happen and this is usually in the first year of life in girls with the less common genetic mutation. Often more than one seizure type is present.

Frequently, girls with Rett syndrome have periods of rapid or slow breathing. These periods are sometimes associated with faints, which can be mistaken for epileptic seizures. Some girls will also have a disturbance of their heart rate and rhythm, the medical name for which is an ‘arrhythmia’.

When boys are affected, the epileptic seizures nearly always start within the first few weeks or months of life and are often extremely difficult to treat.

The discovery of the genetic mutation in Rett syndrome has made it much easier to diagnose the condition. However, it is obviously important for doctors to think about the condition in the first place so that the blood test can then be done to look for the genetic mutation. This common mutation is found in about 80 per cent (eight out of every 10) girls with Rett syndrome. Before the genetic mutation was discovered, the diagnosis of Rett syndrome was not usually made until girls were aged three, four or even six years of age.

The electroencephalogram test (EEG), which measures electrical activity in the brain, may show features which suggest Rett syndrome even when epilepsy is not present, and therefore can be useful in diagnosis.

Brain scans are normal in children with Rett syndrome.

Treatment of this condition can be very difficult. Anti-epileptic drugs that are often used include sodium valproate (Epilim), lamotrigine (Lamictal), carbamazepine (Tegretol, Tegretol Retard) and clobazam (Frisium). Other medications, including piracetam (Nootropil) may help to control seizures in some cases.

Prognosis (Outlook)
The seizures usually become less of a problem in adolescence and early adult life but only occasionally will the seizures stop completely.

With Rett syndrome there is usually increasing difficulty with walking and they may eventually not be able to walk. Many older children and teenagers will need special attention to prevent scoliosis (bending or curvature of the spine).

Feeding may also be a problem, particularly in early childhood and some children will need to be fed through a nasogastric tube (tube inserted through the nose) or even a feeding gastrostomy tube (tube inserted directly into the stomach).

The life-span of people with Rett syndrome is shortened, but most lives into early and middle adulthood. Unfortunately, most boys with Rett Syndrome will have a very shortened life-span.

Finally, all girls with Rett syndrome need complete care throughout their lives.

Support organisations
Rett Syndrome Association UK, Langham House West, Mill Street, Luton, LU1 2NA
tel: 01582 798910, fax: 01582 724129

Molecular Diagnosis of Rett Syndrome (Interesting and must read article by Dr. Peter Huppke)


The possibility of detecting mutations in MECP2 in patients with Rett syndrome has changed the face of this unique disorder and has accelerated research in this field. Several articles have already been written about the genetics of Rett syndrome. In this article, we do not attempt to discuss the genetics of Rett syndrome in great detail but instead summarize knowledge that is important for clinicians caring for these patients and their parents.
Description of the First Mutations in MECP2

It had been speculated for many years that genetic defects in the X chromosome are involved in the pathogenesis of Rett syndrome because almost only female individuals are affected. It was assumed that there is male lethality in this condition. However, because greater than 99% of Rett syndrome cases are sporadic, linkage studies were not possible until 1998, when a family identified with a maternal inheritance pattern of Rett syndrome permitted exclusion mapping studies to be performed that then defined chromosome Xq28 as a candidate region for the Rett syndrome gene.[1] In 1999, Amir and colleagues identified the first mutations in MECP2 in 5 of 21 sporadic cases with Rett syndrome.[2] MECP2 was not one of the first candidate genes on chromosome Xq28 that were investigated because it is expressed in all tissues and had no known brain-specific function.[3]

MECP2 and the MECP2 Protein

The MECP2 gene that is mutated in Rett syndrome is located on the long arm of the X chromosome at Xq28 and is subject to X-chromosome inactivation.[4,5] The gene consists of four exons that code for two different isoforms of the MeCP2 protein. Until early 2004, it was thought that only one isoform existed. For this isoform, a start codon in exon 2 is used and exon 1 is noncoding, explaining why, until recently, exon 1 was not analyzed in the routine mutation screening. However, it has since been found that there is a second isoform of the protein in which exon 1 includes coding sequences and exon 2 is eliminated by alternative splicing (Figure 1). Unexpectedly, this new protein isoform is far more abundant than the one originally described.[6,7] The protein isoform that was described first was subsequently named MECP2e2, and the newly described isoform was named MeCP2e1.

The MeCP2 protein was first described in 1992.[8] It is one of five known proteins sharing a methyl-CpG binding domain that allows them to bind to methylated CpGs in the DNA. Through different mechanisms, this binding leads to a transcriptional repression of genes in the area of these binding sites. The exact mechanisms of this suppression are the topic of other articles in this issue. To understand the significance of the position and type of mutation, it is important to know that there are two, possibly three, domains of the protein that are essential for its function. The methyl-CpG binding domain is encoded by exons 3 and 4 and binds to the methylated DNA (Figure 2).[9] The second important domain is the transcriptional repression domain, which contains binding sites for the corepressor complex and is therefore involved in the repression process.[10] The third domain is located in the C-terminus of the protein and is meant to facilitate the binding both to naked DNA and to the nucleosome core.[11] However, this domain is not yet well defined.

MECP2 and the MECP2 Protein

The MECP2 gene that is mutated in Rett syndrome is located on the long arm of the X chromosome at Xq28 and is subject to X-chromosome inactivation.[4,5] The gene consists of four exons that code for two different isoforms of the MeCP2 protein. Until early 2004, it was thought that only one isoform existed. For this isoform, a start codon in exon 2 is used and exon 1 is noncoding, explaining why, until recently, exon 1 was not analyzed in the routine mutation screening. However, it has since been found that there is a second isoform of the protein in which exon 1 includes coding sequences and exon 2 is eliminated by alternative splicing (Figure 1). Unexpectedly, this new protein isoform is far more abundant than the one originally described.[6,7] The protein isoform that was described first was subsequently named MECP2e2, and the newly described isoform was named MeCP2e1.
The MeCP2 protein was first described in 1992.[8] It is one of five known proteins sharing a methyl-CpG binding domain that allows them to bind to methylated CpGs in the DNA. Through different mechanisms, this binding leads to a transcriptional repression of genes in the area of these binding sites. The exact mechanisms of this suppression are the topic of other articles in this issue. To understand the significance of the position and type of mutation, it is important to know that there are two, possibly three, domains of the protein that are essential for its function. The methyl-CpG binding domain is encoded by exons 3 and 4 and binds to the methylated DNA (Figure 2).[9] The second important domain is the transcriptional repression domain, which contains binding sites for the corepressor complex and is therefore involved in the repression process.[10] The third domain is located in the C-terminus of the protein and is meant to facilitate the binding both to naked DNA and to the nucleosome core.[11] However, this domain is not yet well defined.

Mutation Detection in MECP2

In Rett syndrome, greater than 99% of patient mutations are sporadic. Not surprisingly, more than 200 different MECP2 mutations have been described so far. As in other genetic disorders with such a variety of possible mutations, direct sequencing of the coding and splice-site regions is the method most laboratories use. It is important to note that exon 1 was not initially included in the routine screening program for mutations in MECP2 because it was thought to be noncoding. This has changed since the description of MeCP2e1, the new isoform of MeCP2, but it remains controversial as to how frequent mutations in exon 1 are. Mnatzakanian and coworkers described deletions involving exon 1 in 2 of 19 patients with Rett syndrome for whom sequencing of the other regions had not revealed any mutations, whereas Evans and coworkers did not detect any mutations in 97 patients.[6,12] Despite this, the analysis of exon 1 is considered part of the routine sequencing program for MECP2, performed in all patients with clinically defined Rett syndrome and especially in those for whom previously performed analyses have not revealed any mutations in the other exons.

In a more generalized screening approach for the identification of MECP2 mutations in large groups of patients with nondefined mental retardation or other disorders, such as autism, denaturing high-performance liquid chromatography and single-strand conformational polymorphism analyses have been applied successfully.[13-15] Thistlethwaite and coworkers applied an electronic DNA microchip using serial differential hybridization to detect the eight most frequent MECP2 mutations.[16]

Several methods have been applied to detect gross rearrangements of MECP2, including multiplex ligation-dependent probe amplification, gene dosage analysis by real-time quantitative polymerase chain reaction, and fluorescent in situ hybridization.[17-20] Although it is still unclear which is the most reliable, a routine mutation screen of MECP2 should include one of these methods.

Mutations in MECP2

Thus far, more than 200 mutations in MECP2 have been described.[21] To facilitate genotype-phenotype correlation analyses, the locus-specific International Rett Syndrome Association MECP2 variation database has been established ( Eight MECP2 mutation hot spots are located at CpG dinucleotides, all transitions from cytosine to thymine. They comprise 65% of all mutations.[21] The other MECP2 mutations are less frequent; some have been found only once or twice. Although the mutations can be found in all parts of the gene, there is an obvious pattern in their distribution. Most missense mutations are located in the methyl-CpG binding domain and in the last part of the transcriptional repression domain, whereas the nonsense mutations are located mainly between the methyl-CpG binding and transcriptional repression domains and in the first part of the transcriptional repression domain.

In some cases of Rett syndrome, it is difficult to decide whether the nucleotide change found is of pathogenic significance or if it is only one of the several polymorphisms present in MECP2. For some patients, it is helpful to consult the International Rett Syndrome Association MECP2 variation database to learn whether other patients with Rett syndrome with the same nucleotide change have already been described. For other patients, it is informative to investigate the extended family. However, sometimes it remains unclear whether the nucleotide change explains the clinical picture.

It was noted early that there is a region at the C-terminus that harbors a hot spot for deletions (see Figure 2). The proximal breakpoint of these deletions, which can be found in about 10% of patients, is located in a section with repetitive sequence elements between nucleotides 1050 and 1200.[22] Furthermore, many of the larger rearrangements have one breakpoint in this region.[18] They were detected in 16% of patients with classic Rett Syndrome in whom the sequencing had not revealed any mutations.[18]

In the literature, the reported detection rate for MECP2 mutations in patients with clinically defined Rett syndrome is between 60% and 80%. It is obvious that these rates are influenced by the clinical parameters used to select patients for genetic analysis. In our experience, the detection rate in girls with classic Rett syndrome is much higher than reported: detection approaches 95% if the analysis includes exon 1 and the search for large deletions.

Functional Consequences of Mutations in MECP2

The methyl-CpG binding domain of MeCP2 is responsible for the binding to the methylated DNA. Consequently, mutations affecting the methyl-CpG binding domain can be expected to interfere with this binding. Yusufzai and coworkers have shown that missense mutations in this area indeed impair the selectivity for methylated DNA, whereas one of the few nonsense mutations affecting the methyl-CpG binding domain leads to an inability of the protein to bind to DNA altogether.[23] Truncating mutations affecting the transcriptional repression domain lead to a protein that binds to methylated DNA but fails to repress the transcription.[23] They also found that a truncated protein is being degraded faster than wild-type proteins.

Spectrum of Phenotypes With Mutations in MECP2 in Girls

The detection of mutations in MECP2 enables us to confirm the diagnosis of Rett syndrome in cases that do not fulfill all diagnostic criteria, which is important because the spectrum of phenotypes is wide. For example, some girls with Rett syndrome never learn to roll over, sit, walk, or talk, but others are able to sing songs and run, and they have good hand function.[24] Also, some girls with Rett syndrome are born with a microcephaly at birth or a macrocephaly.[24,25]

The speculation that other disorders are caused by mutations in MECP2 has not been proved conclusive. Although some autism cases have been reported, large studies have ruled out MECP2 mutations as playing a major role in the pathogenesis of autism.[14,26] The same seems to be true for patients with Angelman syndrome. Single cases with mutations in MECP2 have been described, but in larger studies, these mutations could not be confirmed in a large proportion of patients with Angelman syndrome.[27,28]

Boys With Mutations in MECP2

A confounding feature of Rett syndrome is that it almost exclusively affects girls; it was previously thought that Rett syndrome is an X-linked disorder with male lethality. However, there was never evidence of this in families affected with Rett syndrome. Studies in patients with Rett syndrome revealed that mutations are almost exclusively of paternal origin.[29] The mutations in MECP2 are obviously a product of spermatogenesis, because the father provides an X chromosome only to his daughters, not to his sons; therefore, boys are not affected.

To date, about 60 male patients with mutations in MECP2 have been reported, and they can be separated clinically into three groups:

Mutations that are found in female patients with Rett syndrome. They show a severe epileptic encephalopathy with frequent apneas. Most of them have been identified because their sisters had classic Rett syndrome. In these cases, the mutation is passed on maternally, not paternally. This seems to be the phenotype of a male patient with a typical Rett syndrome mutation.[30-34]

Mosaic form mutations or XXY Klinefelter syndrome with mutations found in female patients with Rett syndrome. They clinically resemble female patients with Rett syndrome, indicating that the typical Rett syndrome phenotype can develop when only about 50% of all cells express the defective gene.[31,32,35-38]

Mutations not frequently found in female patients with Rett syndrome. The boys in this group present with symptoms that do not resemble Rett syndrome. Most have unspecific, nonprogressive mental retardation; some present with varying neurologic and psychiatric symptoms.[39-46] Overall, this group of patients is not well defined, and for some mutations, the pathogenetic effect has been questioned.[21]


Until 1999, the diagnosis of Rett syndrome was based solely on the clinical picture because no biochemical or genetic markers were known. Through molecular analyses, we have learned that the clinically defined Rett syndrome diagnosis, based on the established criteria, is very reliable because the vast majority of female patients with classic Rett syndrome have mutations in MECP2. In fact, the mutation detection rate in this group is so high that one could question if mutation analysis of MECP2 should even be performed in a typical case. We have also learned that mutations can be found in girls with a clinically incomplete picture of Rett syndrome. With an increasing number of atypical Rett syndrome cases being reported, clinicians have to ask themselves whether MECP2 analysis should be performed in all patients with undefined mental retardation. However, at least in girls, the data gathered so far do not support that approach. Although there have been single reports of female patients with autism or Angelman syndrome who carry mutations in MECP2, larger studies have not provided evidence that MECP2 mutations play a major role in the pathogenesis of these disorders. Altogether, the spectrum of clinical phenotypes in female patients with mutations in MECP2 is wide; there is no current convincing evidence that these mutations are the primary defect for other neurologic disorders. Therefore, the indication for genetic analysis in female patients can be based on the established diagnostic criteria, keeping in mind that not all of them have to be fulfilled. A clinical checklist should be used to minimize the amount of negative genetic test results.[47]

In boys, MeCP2 mutations seem to be very rare and the clinical phenotype is not well defined. It seems unquestionable that all boys with a Rett syndrome phenotype should be tested for mosaic mutations or an XXY genotype (group 2). It also appears reasonable to test boys with a severe encephalopathy and frequent apneas (group 1). In contrast, we do not have enough data available to search for MECP2 mutations in boys from group 3. The phenotypes described up to now are unspecific and too frequent in the mentally retarded population to allow for sensible mutation screening outside research programs.


Sirianni N, Naidu S, Pereira J, et al. Rett syndrome Confirmation of X-linked dominant inheritance, and localization of the gene to Xq28 1998;63:1552-1558.

Amir RE, Van den Veyver IB, Wan M, et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2 Nat Genet 1999;23:185-188.

Amir R, Dahle EJ, Toriolo D, et al. Candidate gene analysis in Rett syndrome and the identification of 21 SNPs in Xq Am J Med Genet 2000;90:69-71.

Adler DA, Quaderi NA, Brown SD, et al. The X-linked methylated DNA binding protein, MECP2, is subject to X inactivation in the mouse Mamm Genome 1995;06:491-492.

D'Esposito M, Quaderi NA, Ciccodicola A, et al. Isolation, physical mapping, and Northern analysis of the X-linked human gene encoding methyl CpG-binding protein, MECP2 Mamm Genome 1996;07:533-535.

Mnatzakanian GN, Lohi H, Munteanu I, et al. A previously unidentified MECP2 open reading frame defines a new protein isoform relevant to Rett syndrome Nat Genet 2004;36:339-341.

Kriaucionis S, Bird A. The major form of MECP2 has a novel N-terminus generated by alternative splicing Nucleic Acids Res 2004;32:1818-1823.

Lewis JD, Meehan RR, Henzel WJ, et al. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA Cell 1992;69:905-914.

Nan X, Meehan RR, Bird A. Dissection of the methyl-CpG binding domain from the chromosomal protein MECP2 Nucleic Acids Res 1993;21:4886-4892.

Nan X, Campoy FJ, Bird A. MECP2 is a transcriptional repressor with abundant binding sites in genomic chromatin Cell 1997;88:471-481.

Chandler SP, Guschin D, Landsberger N, et al. The methyl-CpG binding transcriptional repressor MECP2 stably associates with nucleosomal DNA Biochemistry 1999;38:7008-7018.

Evans JC, Archer HL, Whatley SD, et al. Variation in exon 1 coding region and promoter of MECP2 in Rett syndrome and controls Eur J Hum Genet 2005;13:124-126.

Shibayama A, Cook EH Jr, Feng J, et al. MECP2 structural and 3'-UTR variants in schizophrenia, autism and other psychiatric diseases. A possible association with autism. Am J Med Genet 2004;128B.50-53.

Beyer KS, Blasi F, Bacchelli E, et al. Mutation analysis of the coding sequence of the MECP2 gene in infantile autism Hum Genet 2002;111:305-309.

Buyse IM, Fang P, Hoon KT, et al. Diagnostic testing for Rett syndrome by DHPLC and direct sequencing analysis of the MECP2 gene Identification of several novel mutations and polymorphisms 2000;67:1428-1436.

Thistlethwaite WA, Moses LM, Hoffbuhr KC, et al. Rapid genotyping of common MECP2 mutations with an electronic DNA microchip using serial differential hybridization J Mol Diagn 2003;05:121-126.

Erlandson A, Samuelsson L, Hagberg B, et al. Multiplex ligation-dependent probe amplification (MLPA) detects large deletions in the MECP2 gene of Swedish Rett syndrome patients Genet Test 2003;07:329-332.

Laccone F, Junemann I, Whatley S, et al. Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome Hum Mutat 2004;23:234-244.

Bourdon V, Philippe C, Grandemenge A, et al. Deletion screening by fluorescence in situ hybridization in Rett syndrome patients Ann Genet 2001;44:191-194.

Ariani F, Mari F, Pescucci C, et al. Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome. Report of one case of MECP2 deletion and one case of MECP2 duplication. Hum Mutat 2004;24:172-177.

Miltenberger-Miltenyi G, Laccone F. Mutations and polymorphisms in the human methyl CpG-binding protein MECP2 Hum Mutat 2003;22:107-115.

Lee SS, Wan M, Francke U. Spectrum of MECP2 mutations in Rett syndrome Brain Dev 2001;23(Suppl 1):S138-S143.

Yusufzai TM, Wolffe AP. Functional consequences of Rett syndrome mutations on human MECP2 Nucleic Acids Res 2000;28:4172-4179.

Huppke P, Held M, Laccone F, et al. The spectrum of phenotypes in females with Rett syndrome Brain Dev 2003;25:346-351.

Oexle K, Thamm-Mucke B, Mayer T, et al. Macrocephalic mental retardation associated with a novel C-terminal MECP2 frameshift deletion Brain Dev 2003;25:346-351.

Lobo-Menendez F, Sossey-Alaoui K, Bell JM, et al. Absence of MECP2 mutations in patients from the South Carolina Autism Project Am J Med Genet B Neuropsychiatr Genet 2003;117:97-101.

Watson P, Black G, Ramsden S, et al. Angelman syndrome phenotype associated with mutations in MECP2, a gene encoding a methyl CpG binding protein J Med Genet 2001;38:224-228.

Hitchins MP, Rickard S, Dhalla F, et al. Investigation of UBE3A and MECP2 in Angelman syndrome
(AS) and patients with features of AS Am J Med Genet A 2004;125:167-172.

Trappe R, Laccone F, Cobilanschi J, et al. MECP2 mutations in sporadic cases of Rett syndrome are almost exclusively of paternal origin Am J Hum Genet 2001;68:1093-1101.

Hoffbuhr K, Devaney JM, LaFleur B, et al. MECP2 mutations in children with and without the phenotype of Rett syndrome Neurology 2001;56:1486-1495.

Villard L, Kpebe A, Cardoso C, et al. Two affected boys in a Rett syndrome family Clinical and molecular findings 2000;55:1188-1193.

Wan M, Lee SS, Zhang X, et al. Rett syndrome and beyond Recurrent spontaneous and familial MECP2 mutations at CpG hotspots 1999;65:1520-1529.

Zeev BB, Yaron Y, Schanen NC, et al. Rett syndrome. Clinical manifestations in males with MECP2 mutations. J Child Neurol 2002;17:20-24.

Lynch SA, Whatley SD, Ramesh V, et al. Sporadic case of fatal encephalopathy with neonatal onset associated with a T158M missense mutation in MECP2 Arch Dis Child Fetal Neonatal Ed 2003;88:F250-F252.

Leonard H, Silberstein J, Falk R, et al. Occurrence of Rett syndrome in boys J Child Neurol 2001;16:333-338.

Clayton-Smith J, Watson P, Ramsden S, et al. Somatic mutation in MECP2 as a non-fatal neurodevelopmental disorder in males Lancet 2000;356:830-832.

Schwartzman JS, Bernardino A, Nishimura A, et al. Rett syndrome in a boy with a 47,XXY karyotype confirmed by a rare mutation in the MECP2 gene Neuropediatrics 2001;32:162-164.

Kleefstra T, Yntema HG, Nillesen WM, et al. MECP2 analysis in mentally retarded patients. Implications for routine DNA diagnostics. Eur J Hum Genet 2004;12:24-28.

Couvert P, Bienvenu T, Aquaviva C, et al. MECP2 is highly mutated in X-linked mental retardation Hum Mol Genet 2001; 10:941-946.

Imessaoudene B, Bonnefont JP, Royer G, et al. MECP2 mutation in non-fatal, non-progressive encephalopathy in a male J Med Genet 2001;38:171-174.

Klauck SM, Lindsay S, Beyer KS, et al. A mutation hot spot for nonspecific X-linked mental retardation in the MECP2 gene causes the PPM-X syndrome Am J Hum Genet 2002;70:1034-1037.

Orrico A, Lam C, Galli L, et al. MECP2 mutation in male patients with non-specific X-linked mental retardation FEBS Lett 2000;481:285-288.

Winnepenninckx B, Errijgers V, Hayez-Delatte F, et al. Identification of a family with nonspecific mental retardation (MRX79) with the A140V mutation in the MECP2 gene. Is there a need for routine screening?. Hum Mutat 2002;20:249-252.

Yntema HG, Oudakker AR, Kleefstra T, et al. In-frame deletion in MECP2 causes mild nonspecific mental retardation Am J Med Genet 2002;107:81-83.

Gomot M, Gendrot C, Verloes A, et al. MECP2 gene mutations in non-syndromic X-linked mental retardation. Phenotype-genotype correlation. Am J Med Genet A 2003;123:129-139.

Moog U, Smeets EE, van Roozendaal KE, et al. Neurodevelopmental disorders in males related to the gene causing Rett syndrome in females (MECP2) Eur J Paediatr Neurol 2003; 7:5-12.

Huppke P, Kohler K, Laccone F, et al. Indication for genetic testing. A checklist for Rett syndrome. J Pediatr 2003; 142:332-335.

Rett Syndrome: One Mutation Affects a Cast of Thousands

Rett syndrome arises in early childhood and robs affected children of the ability to speak and control their movements. The disorder is caused by a mutation in a single gene, but scientists have not understood how that mutation disrupts neurological function. Now, Howard Hughes Medical Institute (HHMI) scientists have discovered that the mutation interferes with the regulation of 2,500 other genes.

The researchers said their discovery offers a promising pathway for understanding the broad and variable symptoms of Rett syndrome, which include language and growth retardation, breathing problems, seizures, motor dysfunction, hand-wringing and social impairment.

“The duplication syndrome and Rett syndrome may share many clinical symptoms, but on the level of the neuron they are totally different, and so the treatment would be totally different.”
Huda Y. Zoghbi

Huda Y. Zoghbi, an HHMI investigator at Baylor College of Medicine, Maria Chahrour, a graduate student in Zoghbi's lab, and their colleagues reported their findings May 30, 2008, in the journal Science.

“Rett syndrome is a devastating disease,” says Zoghbi. “Think about what it takes to function normally: You have to be coordinated, you must be able to think, you have to be able to communicate, and you need to move smoothly and with balance.” The symptoms of other neurological diseases affect some of these functions, she says, but Rett syndrome affects them all.

Zoghbi started her career as a pediatric neurologist, and became fascinated by Rett syndrome during her training. “It's very hard not to be intrigued by this disease,” she says. “We all are familiar with neurological diseases; I'm sure everyone has seen some one with epilepsy, or Parkinson's disease, or bipolar disorder or schizophrenia. But when you see symptoms of all these diseases in one individual, you are struck by this.” Rett syndrome is particularly heartbreaking, she says, because it develops after the child has already learned to walk, and perhaps even say a few words. That's part of what drove her to study it. “To lose all that, and gradually develop a symptom from almost every neurological disease in the book is quite mind boggling,” she says.

In 1999, Zoghbi's group showed that a mutation in the MECP2 gene causes Rett syndrome, which affects one in about 10,000 girls. The MECP2 gene resides on the far end of the longest arm of the X chromosome. During development, certain genes that are to be silenced, or prevented from being expressed, are physically marked by the addition of methyl groups. MECP2 encodes a protein named methyl-CpG-binding protein 2, or MeCP2, that binds to these methylated genes. Once bound, MeCP2 attracts other proteins that prevent access to the cell's DNA transcription machinery, thereby facilitating gene silencing.

Zoghbi and other researchers have worked to understand how a single genetic mutation can be responsible for Rett syndrome's many symptoms, but they have met with little success. “The protein was known to orchestrate gene expression in neurons,” she says, but the question that remained was “What genes is this regulator talking to?”

Recently researchers discovered a mutation that adds a duplicate copy of MECP2 to the genome. This doubles the amount of the MeCP2 protein in cells rather than eliminating it, as in Rett syndrome -- but otherwise causes similar symptoms. The human MECP2 duplication syndrome was described so recently that no one knows how common it is, or why having too much of the protein is just as harmful as not having it at all.

As researchers search for the genes that are regulated by MeCP2, they typically compare gene activity patterns in the brains of mice that lack the protein to patterns in the brains of normal mice. Those kinds of studies, Zoghbi says, have identified changes in the activity in a handful of genes, or even nothing at all—not the sort of widespread disruption that one might expect to be responsible for the variety of symptoms associated with Rett syndrome.

Zoghbi thought researchers might be getting those results because they were casting their nets too wide. As a result, they were not seeing changes because they were lost against the vast backdrop of gene activity in the whole brain. So, she took an educated guess and narrowed her search to a small area of the brain called the hypothalamus. In humans, the hypothalamus is about the size of an almond. In mice, it's no larger than a split pea. “It's responsible for many metabolic processes,” says Zoghbi. “When you get thirsty, there's a hormone produced in the hypothalamus that tells you to drink water. It controls how much stress hormones you have; when it's time to shiver because you're cold. It's a command center.” With its broad purview, Zoghbi says, it's reasonable that misregulation within the hypothalamus could be responsible for many of the symptoms of Rett syndrome

Zoghbi measured gene expression in the hypothalami of normal mice, as well as mice with Rett syndrome or the MECP2 duplication syndrome to increase the likelihood of finding MeCP2 targets. She found that the disorders changed the expression of about 2,500 genes. In the animals with Rett syndrome, 2,200 were less active than they were in normal mice, while the remainder showed increased activity. The numbers were precisely reversed in the duplication syndrome. These findings were surprising because they show that MeCP2 can activate genes.

“From a practical viewpoint, knowing how the two syndromes behave on a molecular level is really important,” said Zoghbi. “The duplication syndrome and Rett syndrome may share many clinical symptoms, but on the level of the neuron they are totally different, and so the treatment would be totally different.”

In addition, knowing just how many genes are affected is important in and of itself. If only a few genes were affected, she says, it might be possible to artificially control those proteins directly. As it is, “we're going to have to find a way to co-opt another master regulator to fill in the gaps,” she says.

Source: HHMI News

Sunday, November 8, 2009