Vienna RTT 50.1 – Full Report

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By Danijela Szili.

RTT 50.1 Vienna 15-17 September

This was not my first Rett conference but was a very special one. It was not just about scientific sessions or sessions about clinical issues and communication but also about history of Rett syndrome. We witnessed many wonderful speeches from physicians, scientists and parents who were here from the beginning and to whom we are thankful for what has been achieved today.

The Vienna congress was about very dedicated people who became also emotionally attached to Rett individuals. For them, Rett research is not just about mouse models and molecules. It is about our girls and women who are charming and have adorable personalities or very strong characters and make people fall in love with them and fight for them. Above all this was a conference about Andreas Rett and his observation in 1964 that started this long journey. His colleagues from around the world talked about his charismatic personality and big influence he had in Vienna not only in his medical work but also in the fight for the Rett people and other disabled individuals in Austria. Thanks to him, a department for individuals with neurological syndromes was created in a clinic in Vienna. Patients finally started having a proper medical care, were not seen any more in residential homes or institutes for the elderly people. His passion for Rett Syndrome was extraordinary and he helped parents to start the first Rett Associations and Foundations in Europe and the USA.

The first talk about the work of Dr Andreas Rett was given by a young colleague of him at that time Germain Weber,Ph.D., professor of Psychology at the University of Vienna, Department of Health, Development and Intervention. The title of his talk was: On the origins and rise of Rett syndrome

Dr. Weber’s research is focusing on health and mental health issues both in people with intellectual and developmental disability. He started working with Dr Andreas Rett in 1984. These were very exciting times for Rett syndrome when the international agreement on the diagnostic criteria was established during the symposium on Rett syndrome in September in Vienna with proceedings published in 1985.

However, what is nowadays known as Rett syndrome had been described as early as 1965 by Andreas Rett with the original publication in 1966 followed in the same year by a short contribution in a Viennese medical journal published in German language. This early description offers almost the complete clinical symptoms nowadays recognized as diagnostic criteria for Rett syndrome and its full title translated in English is: On remarkable syndrome of cerebral atrophy associated with hyperammonaemia in childhood. The fact that it was published in German and had hyperammonaemia being the first characteristic of the syndrome probably caused this twenty years of incubation period and the fact that his work was not accepted within the Viennese medical research community and stayed unknown outside Austria. Shortly after the original publication the hyperammonaemia hypotheses turned out to be caused by a sloppy lab work but this mistake already had its consequences and Dr Andreas Rett’s work was strongly contested in the medical community. This did not stop him to believe in his theory and to continue fighting for his cause.

According to Rett’s personal account, it occurred in 1964 when one day he walked in the crowded waiting room in his hospital and saw two little girls side by side on their mothers’ laps , rocking slightly with distant look in otherwise impressive eyes. What struck him were their hands in frantic movement. He searched for medical books and could find no description of this. He called his assistant, nurse Martha mentioning that there might be other cases like this and she found 22 of them who were then included in the study that resulted with the previously mentioned article published in 1966. He also made a film at the beginning of this unusual odyssey to bring to the world’s attention to what he had discovered.

Bengt Hagberg, a leading Swedish researcher in the field of neuropaediatrics had studied the similar cases and was on the way to publish these when he met Dr Andreas Rett during the Toronto conference in 1982. Dr Hagberg lectured on the cases he had studied, brought up his theories from the yet unpublished paper in which he referred to the hand-washing syndrome. Rett addressed him and invited Hagberg to Vienna to evaluate his cases. In the meantime the Hagberg’s international team recalled their manuscript, reviewed it, to be finally published in 1983 in the Annals of Neurology which reported the cases with the label Rett syndrome. The full title of this publication was: A progressive syndrome of autism, dementia, ataxia and loss of purposeful hand use in girls: Rett syndrome. It was a report of 35 cases. Among these cases were Rett girls from France and Portugal. Authors were Bengt Hagberg, Jean Aicardi, Karin Dias, Ovidio Ramos.

Hagberg’s acknowledgement of Rett’s pioneering first description is a stunning gesture and an expression of a noble character showing deep academic appreciation of Rett’s early contribution. This was the beginning of a series of international conferences on Rett syndrome, taking place in Vienna, the US and Japan.

Besides the rising commitment of many researchers from various fields to contribute to the enigma of Rett syndrome the evolution on the syndrome’s knowledge was strongly supported and influenced by the activities of the International Rett Syndrome Association founded in 1984 by Kathy Hunter and few other parents in the USA. This was followed by foundation of a Rett association in UK (Rett UK) by Yvonne Milne in 1985. These two extraordinary Rett mothers were in Vienna and both gave very emotional speeches about their work and friendship with Andreas Rett.

The next speaker was Prof. Dr. Folker A. Hanefeld from the Department of Paediatrics and Paediatric Neurology, Faculty of Medicine, Göttingen (Germany).

He explained why was Dr Andreas Rett’s first publication on Rett syndrome not a success. Its title had two weaknesses: it was written in German, rather than English and it was based on the test that had gone wrong. The hyperammonaemia found in these girls could not be confirmed by others. Rett’s ingenious discovery was heading in the wrong direction. Patients who fitted the clinical diagnoses failed to meet the biochemical aspect. It was the time of metabolic screening and “No metabolic defect, no new disease” was a doctrine at the time.

Prof Hanefeld was present at the symposium in Vienna in 1984, organized by Andreas Rett and Bengt Hagberg, where the inclusion and exclusion criteria for the diagnosis of Rett syndrome were formulated.

Dr Hanefeld and Bengt Hagberg were also very good friends and they met regularly to discuss their Rett cases.

In this talk the Baltimore meeting, organised by Dr Moser at the John Hopkins Medical University in 1985 was mentioned where Rett Syndrome was discussed for the first time in US. Drs Alan Percy, Huda Zoghbi, Andreas Rett and Bengt Hagberg were also there, together with many Rett girls. At this meeting Dr Hanefeld met child psychiatrist Michele Zappella from Sienna. Atypical Rett syndrome variant with a mild phenotype also called preserved speech or “forme fruste” variant carries his name.

Atypical Rett variant Early Seizure variant caused by mutations on CDKL5 gene is sometimes mentioned in scientific literature as Hanefeld variant.

 

Dr Yoshiko Nomura, Neurological Clinic for Children, Tokyo, Japan.

Dr Nomura talked about her relation to Prof. Rett and how he very much influenced her and Dr Segawa’s work. They met for the first time in 1982 and at that time Dr Nomura already had four patients with the same symptoms. They worked together with Andreas Rett and met on a regular base. In March 1988, Andreas Rett visited the Segawa Neurological Clinic for Children. For that occasion Dr Nomura and Dr Segawa organized a symposium for many Japanese clinicians and researchers. Prof. Rett also examined the patients there. Two Japanese clinicians have been doing research about sleep abnormalities and locomotion in Rett syndrome.

 

Synaptic abnormalities in RTT and their impact on brain plasticity was the next topic. Dr Michael Johnston (Baltimore, John Hopkins University School of Medicine and Kennedy Krieger Institute) presented the overview about the neurotransmitters (glutamate and GABA) levels abnormalities in the post mortem cortex of Rett syndrome patients and in a mouse models and a clinical trial of dextromethorphan (DEX) as an intervention.

A number of clinical (seizures, agitation, cognitive impairment, sleep abnormalities) and pathological (declining brain growth) abnormalities pointed to a disorder of synapse formation and regulation in RTT even before the MeCP2 gene was discovered. Synaptic counts in post-mortem brain tissues from RTT individuals have been shown an approximate 50% reduction in synapse numbers as was found in a RTT mouse model.

In 2009 the group led by Dr Sakkubai Naidu reported that brain levels of glutamate (main excitatory neurotransmitter) in younger girls with Rett was higher than in the control group compared to the older girls who had lower levels of glutamate compared to age-matched controls.

Recently his team monitored the 24 hour continuous sleep-wake cycle in the Bird model of the Mecp2 knockout mice and elevated levels of glutamate were 40% higher than normal controls. These studies showed that abnormal sleep-wake cycles in the mice and long periods of sleeplessness were associated with very high levels of glutamate in the brain.

A number of papers have also shown changes in GABAergic inhibitory neurons in post-mortem brain tissue from Rett patients and in animal models.

This suggests that the cognitive disorder and some other issues like seizures could be improved with interventions leading to a reduction of NMDA (one of the glutamate receptors) mediated activity and/or enhancing GABAergic activity specially if applied early in life. This was the reasoning behind the clinical trials of dextromethorphan (DEX). DEX is an approved OTC (over the counter) cough suppressant which is a competitive inhibitor of the NMDA receptor. Dr Sakku Bai Naidu is a principal investigator for these two clinical trials. The second one, the fully blinded Placebo Controlled Trial (https://clinicaltrials.gov/ct2/show/NCT01520363?term=rett&rank=23) is still ongoing but it is expected that the final subject (patient) will enter in the next month. The first one, the Open Label study (https://clinicaltrials.gov/ct2/show/NCT00593957?term=rett&rank=21) was terminated due to FDA requirement to change the design to the fully blinded, placebo controlled trial and start a new phase II trial.

 

Progress in Rett syndrome: first steps to natural history study. Alan K. Percy. University of Alabama at Birmingham (USA).

In the USA, in the early 2000s the Rare Disease Clinical Research Network (https://www.rarediseasesnetwork.org/) was formed and initial funding for the natural history study (NHS) became available. The RDCRN is designed to advance medical research on rare diseases by providing support for clinical studies and facilitating collaboration, study enrolment and data sharing. Through the RDCRN consortia, physician scientists and their multidisciplinary teams work together with patient advocacy groups to study more than 200 rare diseases at sites across the US.

Natural history study regarding RTT was refunded twice by RDCRN and now in its third iteration is addressing RTT, MECP2 duplication disorders, and other RTT-related disorders including CDKL5, FOXG1 and individuals with MECP2 mutations, both females and males, who do not meet the diagnostic criteria for RTT. Until now data from more than 1200 participants in the first 11 years of the study have been evaluated and published. The current study is continuing to collect information on the national history of the above mentioned disorders, collecting biologic samples for possible detection of a biomarker, and investigating the event-related potentials of hearing and vision (auditory brainstem and visual evoked potentials).

One of the outcomes of the NHS was validation of the revised diagnostic criteria of 2010. An international panel of clinicians simplified the criteria from 2002 and provided a distinction for their application in classic and variant or atypical RTT.

Neul JL., et al. Rett syndrome: revised diagnostic criteria and nomenclature. Ann Neurol. 2010

Percy AK, et al. Rett syndrome diagnostic criteria: lessons from the Natural History Study. Ann Neurol. 2010

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3021984/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058521/

As we all know, RTT is a multisystem problem and in addition to brain growth, stereotypies, epilepsy, periodic breathing, sleep and other problems directly connected to the Central Nervous System (CNS), many other systems are affected, including growth and nutrition, the Gastro Intestinal Tract (GIT) and pubertal development.

Longevity is reduced by about one third compared to typically developing females. The good news is that, thanks to the better care and interventions, the survivor rate compared to the original group described by Andreas Rett has at least doubled. This is related to improved diagnosis, nutrition, physical and occupational therapies (importance of preserved ambulation or assisted walking) and effective seizure control. Death was often unwitnessed and causes were difficult to determine, but cardiorespiratory issues were regarded as the most likely cause of difficulties.

Prolonged QTc interval is present in approximately 20% of individuals in the NHS. It is usually in the borderline range and the parents should be careful with the medicinal products their daughter is taking. Some medicinal products tend to prolong the QTc interval. This list is updated very often and can be checked on http://www.rettsyndrome.eu/wp-content/uploads/2015/10/CombinedList.pdf.

Occasionally, QTc is sufficiently elevated and needs treatment by β-blockers. Rarely, QTc levels exceed 600ms, in which case the pacemaker has been placed. McCauley et al. demonstrated similar QTc prolongation in an animal model of RTT and found that antiarrhythmic drugs, Na channel antagonists (blockers) like were more effective than β- blockers.

Early development had been characterized as “apparently “ normal but thanks to the studies of Einspieler and Marschik taking advantage of video and audio recordings during infancy, substantial deviations from normal were noted. Data from the NHS confirmed these findings.

About Growth. Declining growth rates are typical. First is the declining head circumference growth rate which could be a very good early diagnostic tool.

Tarquinio DC, et al. Growth failure and outcome in Rett syndrome: specific growth references. Neurology 2012

 

Gastrointestinal features.

Problems involving the GIT are seen from top to bottom and deserve primary attention when the individual is upset or unhappy, or awakens frequently in the night. Gastrostomy tube placement provides some or exclusive feeding in more than 30%.

Motil KJ, et al. Gastrointestinal and nutritional problems occur frequently throughout life in girls and women with Rett syndrome 2009

https://www.ncbi.nlm.nih.gov/pubmed/22331013

Leonard H, et al. Assessment and management of nutrition and growth in Rett syndrome

2013

https://www.ncbi.nlm.nih.gov/pubmed/24084372

Baikie G, et al. Gastrointestinal dysmotility in Rett syndrome 2014

https://www.ncbi.nlm.nih.gov/pubmed/24121144

Puberty and menarche generally occur similar to typically developing females, but important differences do exist.

Kilian JT, et al. Pubertal development in Rett syndrome deviates from typical females. 2014

https://www.ncbi.nlm.nih.gov/pubmed/25283752

Clinical trials.

Dr Alan Percy mentioned IGF-1 clinical trial in Boston Children’s hospital and NNZ-2566 (Trofinetide) the terminal tripeptide of IGF-1, at Baylor College of Medicine, UAB, and Gillette Children’s Hospital. Both agents proved to have no significant safety or tolerability issues. The IGF-1 trial produced evidence of safety and is continuing at multiple sites. The Trofinetide trial in older girls and women provided preliminary evidence of efficacy and second phase 2 trial is being conducted in children aged 5-15.

Other two trials are with Sarizotan and Ketamine.

Sarizotan, a serotonin and dopamine receptor agonist will be tested in girls and women aged 13-50 in several sites in US, India and Italy and will target breathing issues, specially apnoea and hyperventilation.

Ketamin trial is open for recruitment in Cleveland Clinic. Ketamin is already approved and used in paediatric population for total anaesthesia. Rational behind the trial is explained in the publication:

Kron M, et al. Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network that are reversed with ketamine treatment 2012

The goal of this trial is to determine the safety and tolerance in children with Rett Syndrome. This study will also evaluate whether the respiratory and behavioural symptoms of Rett Syndrome are improved with Ketamine treatment.

 

Clinical and genetic diagnostic criteria in Rett syndrome Jeffrey Lorenz Neul, Department of Neurosciences, University of California, San Diego.

Although the vast majority (95%) of people with typical Rett syndrome have disease-causing mutations in MECP2 gene clinical diagnoses or definition of disease is more important.

The first important issue that reinforces the importance of the clinical diagnosis is the fact that some people who clearly have the clinical features of Rett syndrome but do not have any identified mutations in MECP2.

In some of the atypical forms of Rett syndrome, such as the Early Seizure Variant or the Congenital Variant, mutations in different genes have been identified. Specifically, mutations in the gene CDKL5 have been associated with the Early Seizure Variant and mutations in the FOXG1 gene have been associated with the Congenital Variant.

Additional genetic studies on people with clinical RTT but lacking MECP2 mutations have found mutations in a number of other genes like for example another gene on the X-chromosome known as WDR45, which is known to be associated with BPAN syndrome. http://nbiacure.org/our-research/in-the-clinic/bpan-rett-project/

Interestingly, the genes mutated in these people with features of clinical Rett syndrome are also mutated in other neurodevelopmental disorders and in epilepsy syndrome.

https://www.ncbi.nlm.nih.gov/pubmed/25914188

https://www.ncbi.nlm.nih.gov/pubmed/25714420

The case described in this study suggests a relationship between the Rett syndrome and the STXBP1 gene not described so far, making the search for STXBP1 gene mutations advisable in patients with Rett syndrome and early onset of epilepsy.

Some patients with Rett features and without MECP2 mutations have been found to have a mutation on TCF4 gene and Pitt Hopkins syndrome: https://pitthopkins.org/what-is-pitt-hopkins-syndrome/

or PURA syndrome: http://www.purasyndrome.org/

The second important issue that emphasizes the importance of a clinical definition of Rett syndrome over a genetic definition is the identification of people with mutations in MECP2 who do not have Rett syndrome. The fact that people can have mutations in MECP2 and be unaffected, have a markedly more severe and distinct disease course, or not show characteristic features of Rett syndrome (but have juvenile schizophrenia for example) indicate that a MECP2 mutation is not sufficient for the diagnosis of Rett syndrome.

Taking all this into account, in 2010 an international group, RettSearch Consortium (http://rettsearch.org/) set out to create revised clinical criteria for the diagnosis of typical and atypical Rett syndrome emphasizing the clinical nature of the diagnosis. This revised criterion has been widely adopted in the field and is utilized for clinical trials as well as clinical care.

Dr Neul talked about some of the interesting results of the NHS considering biomarkers. 226 participants were analysed and 35 biochemical compounds had very significant up or down values in comparison with normal values. Most of those had a role in lipid and energy metabolism. One of those was amino acid tryptophan which is precursor in the synthesis of serotonin and other neurotransmitters from the monoamines group.

Behavioural Biomarkers of typical Rett syndrome: moving towards early identification.

Christa Einspieler.

Christa analysed home videos and data from questionnaires filled in by parents. Videos were showing babies before 6 months who were videoed by their parents before they got the diagnosis of Rett syndrome. All the babies had typical Rett syndrome and mutations on MECP2 gene (R168X, R255X). The videos were also analysed by a group of experts in child development who did not know if the baby they are watching had Rett syndrome or not.

They found out that general movements were abnormal in all 22 individuals later diagnosed with RTT. Among other behavioural abnormalities tongue protrusion (68%), postural abnormalities (58%), asymmetric opening of the eye lid after a blink (56%), first hand stereotypies (42%), or bizarre smiling (32%) were the most frequently occurring atypical behaviours. Hand stereotypies included grasping movements in the fingers and peculiar wrist movements.

Together with Peter Marschik, who is trained in neurolinguistics, these with new family videos were re-analysed and a number of peculiarities in early verbal behaviour of infants and toddlers later diagnosed with RTT were found. Those infants pass developmental tests since they did coo and babble.

Other early signs were frequently reported like early feeding difficulties and insensitivity to pain.

 

The challenging RTT: to watch, to measure and to compare Eric EJ Smeets

Rett Expertise Centre-Maastricht, Netherlands.

The clinical expertise in the Rett Centre is a result of many encounters with girls and women with RTT (more than 400 individual cases and 60 cases in follow up).

Thanks to the development of the modern techniques in molecular genetics we now know a lot of detectable mutations and rearrangements in the MECP2 gene confirming the clinical diagnosis of the typical RTT in the vast majority of cases (95-97%). Whole exome sequencing leads also to a number of atypical MECP2 related phenotypes in girls (and boys) with learning disabilities, that were not suspected clinically and to newly discovered pathogenic mutations in other genes (e.g. CDKL5, FOXG1, TCF4, PURA).

Genotype phenotype correlation studies pinpointed to those mutations with better prognosis and outcome, like for example CTS deletions and R133C. Nevertheless variation in the clinical severity of RTT is large and the search for the epigenetic regulatory factors is on-going.

CTS deletions C terminal hot spot deletions.

Link to article

Brainstem immaturity in RTT is a longstanding issue and causes periodic breading, non-epileptic episodes, sleeping issues, heart rate abnormalities, GIT dysmotility and sometimes urinary retention. In the Maastricht Center is done a cortical-bulbar neurophysiologic assessment in order to monitor breathing irregularities and discriminate epileptic from non-epileptic spells in Rett individuals.

In this assessment all these features are monitored at the same time: breathing, heart rate, blood pressure and gasses, cardiac vagal tone, baroreceptor sensitivity. Video EEG during the daytime and sleep recordings are also performed. All the collected data is then evaluated by a multidisciplinary commission. After the discussion the conclusion is presented to parents.

His take-home message is that Rett syndrome patients are over treated with antiepileptic drugs.

Epilepsy is present in only 1/3 of assessed patients and others had rett-like episodes caused by the brainstem immaturity and dysautonomia (abnormal functioning of the autonomic nervous system). Some of these non-seizure events were stiffening, pupil dilatation, involuntary movements…

In recent multicentre study they used objective and robust data of cardiorespiratory variables in the investigation of the genotype-phenotype correlation in RTT. All females had dysautonomia, and this was not restricted to, nor influenced by, one specific group or single recurrent mutation.

In the centre a large focus on communication is also experimented, using eye gaze technology in developmental testing, neurocognitive profiling, training in communicative ability in general and emerging literacy in the younger girls. Gillian Townend is communication specialist in the Maastricht Rett Centre and was also present at the congress.

The focus is also on ageing in RTT, which is observed thanks to the longitudinal study that is still going on and has a special focus on the age group above 35 years and a smaller survivor group above 50 years.

MeCP2 protein regulates gene expression of many other genes and it up and down regulates metabolites in RTT. Finding the right target genes/proteins/metabolites may take us to the known drugs which interfere with that exact pathway leading to improvement of symptoms (lovastatin clinical trial in the USA is an example). Bioinformatics help us in this. Friederike Ehrhart who is working for the centre presented their work in: New insights in Rett syndrome using pathway analysis for transcriptomics data.

Transcriptome is the set of all messenger RNA molecules in one cell or a population of cells and it reflects the genes that are being actively expressed at any given time.

Pathway analysis is done with software like PathVisio and combines experimental transcriptome data with structured existing knowledge. Bioinformatics analysis and visualization methods and tools allow explanation and proof of whether and how a certain biological pathway is altered as a consequence of the experiment, which maybe a mutation, drug application, nutrition, or disease.

In this study Friederike and her team investigated the differentially expressed genes and affected pathways in four different primary neuronal cell types using published data from a Mecp2 mouse model originally published by Sugino et al.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Cell-type-specific+represion+by+methyl-CpG-binding+protein+2+is+biased+toward+long+genes

The original study by Sugino et al. already indicated that each of the four investigated subpopulations of neuronal cells of a mouse model expresses a different set of genes in comparison to wildtype, and they found especially cell adhesion genes and long genes to be overrepresented among the changed genes. In the present study the raw transcriptomics data were reanalysed using ArrayAnalysis.org. Finally, lists of differentially expressed genes were extracted and pathway and gene ontology term enrichment analysis was performed to reveal the biological pathways changed in this model system.

258 genes are differentially expressed in Purkinje cells, 850 in locus coeruleus neurons, 463 in the pyramidal neurons in the motor cortex (TTL5) and 301 in fast spiking interneurons of the motor cortex.

Pathway analysis showed that fatty acid metabolism pathways are predominantly modified in fast-spiking interneurons of the motor cortex. In the locus coeruleus neurons (located in the brainstem), glutathione and amino acid metabolism is affected, in which the expression of glutathione synthetase and glutamate-cysteine ligase is significantly downregulated. Both enzymes are directly involved in the production of glutathione out of glutamate.

Gene ontology term enrichment analysis showed that glutamate binding and glutamate receptor activity was changed in Purkinje cells (GABAergic neurons) and TTL5 while ion (Ca₂₊ and K₊) flux-related receptors, channels, and binding proteins changed in all cell types.

For Purkinje cells and fast-spiking interneurons one of the most changed processes is regulation of the cholesterol metabolism.

A more detailed investigation of the data is necessary, especially to separate between up- and downregulated processes and to link them to their specific function.

 

Helen Leonard

Databases today and tomorrow: relevance for clinics and research.

InterRett-International Rett Syndrome Study is funded by rettsyndrome.org

The Australian Rett Syndrome Database (ARSD) was established in 1993 to investigate this relatively newly described disorder for which, at that time, there was no known cause. The call for this research came from the family association: the Australian Rett Syndrome Association.

Following initial data collection from families there have been successive waves of data collection from families every two to four years since 1996, and this data have been supplemented with contribution from clinicians and hospital records. The ARSD is therefore population-based and longitudinal.

There are currently more than 400 girls and women registered, approximately 18% have died. The goals of ARSD are to investigate the natural history of Rett syndrome and after the discovery of the MECP2 gene, to explore relationships between genotype and phenotype.

Early concerns

Parents participating in the study have observed subtle developmental deviations. This finding from the study published in 1998 led to a change in the criteria in 2002, such that the early development was “apparently “ normal and that there might be subtle or more overt abnormalities in the early months of life. These reported observations were later validated in studies of home videos, where many infants demonstrated altered gross motor and communication function.

Crawling appears to be a big challenge for children with RTT. As for walking there are relationships with genotype. The walking is more likely to be learned by girls with mutations associated with a milder clinical phenotype such as R133C, R306C or a C-terminal deletion.

Regression

Regression of communication and hand function skills is a fundamental feature of RTT and can occur rapidly or more gradually over a longer period of time. At regression the child may withdraw socially and often cry inconsolably, sometimes for many months. The development of impaired gait may also become more apparent, and loss of balance in sitting, standing and walking. GIT issues and sleep disturbances can became apparent, as can other subtle signs like strabismus or altered postures.

The challenges of diagnosis

Getting the diagnosis might be a challenge for some even in well developed countries like Australia. In milder cases when the clinical picture does not really match with the original criteria the clinicians might be reluctant to do the genetic testing and think it is not considered appropriate or cost-effective. There is a need for clinicians to be aware of the variability in presentation.

Comorbidities

RTT is associated with a high prevalence of comorbidities, many of which have been featured as supportive diagnostic criteria. These include epilepsy, scoliosis, poor growth and gastrointestinal problems, unusual breathing patterns (periodic breathing), difficulties with sleep and a high risk for bone fractures.

Some antiepileptic drugs are increasing the risk of bone fractures in Rett individuals. These are valproate, carbamazepine, and lamotrigine. Progesterone medication also increases the probability of fractures. Some anti-reflux medication should also be taken with caution (rabeprazole) since they can make a risk of fractures higher if taken for one year or more.

Survival rate in Rett individuals is higher if they have had a spinal surgery. After the gastrostomy button placement the Body Mass Index (BMI) scores increased by 0.89.

The evidence base for each of these comorbidities in RETT is generally poor, and clinicians depend heavily on the clinical experiences of experts and management principles applied to other children with developmental disability. Three sets of guidelines have been developed to support the clinical management of scoliosis, gastrointestinal health and bone health in RTT.

http://www.aussierett.org.au/resources/guidelines,-reports-and-books/

Although their level of evidence is low, these guidelines provide important baseline information for clinicians and therapists.

Jenny Downs is a physiotherapist and researcher who has been working with Helen Leonard at the Telethon Kids Institute in Perth for more than 11 years.

She talked about Gross motor function and activity in Rett syndrome.

As a part of the Australian Rett Syndrome Study they have collected video data in 2004, 2007 and 2012 to observe functional abilities including gross motor skills in girls and women with RETT across Australia. 255 were collected. These observations were the base for the development of the 15-item Rett Syndrome Gross Motor Scale (RSGMS) and also to assess its measurement properties.

One of the conclusions was that overall across childhood and in adult years; the distribution of skills broadly reflects those of the early years. The majority remain able to sit, slightly fewer than half walk independently and a smaller proportion can perform transition movements. Strong relationship between motor skills and mutation type has also been demonstrated and that provide additional validation of the scale. The RSGMS potentially has a role to play in clinical monitoring and as an outcome measure in clinical trials.

The second part of the study was to see how these gross motor skills are used and to measure time spent active, number of steps walked. It was found that StepWatchActivityMonitor (SAM) accelerometer was accurate in measuring steps. SAM was put at the girls’ ankle and was used to assess whole day activity in 64 girls and women, three quarters walked independently and one quarter with assistance. Mothers’ diary cards were also used to quantify up (standing and walking) and downtime (lying and sitting).

The median average daily steps was 5093 steps (range 458-32,835) and the median percentage of sedentary time was 60% (range 18-94) indicating that activity in RTT is generally reduced but that there is also a marked variability. Those who walked independently were more active but walking was mostly slow with less than 20 steps in a minute for both groups. Scoliosis and epilepsy managed with polytherapy (therapy with two or more medications) were associated with less physical activity and more sedentary time. The increase in sedentary time and less uptime in the teenage years occurred before the marked deterioration in steps in adulthood and this could indicate that negative environmental influence during the teenage years had an important role to play. Assisted walking is very important for the overall health of the Rett individual but specifically for: smaller risk of pneumonia, social contacts, doing things and going out is fun, communication skills, in day to day care and management. Therapy, activity and quality of life of teenage girls should be addressed more seriously since this is the critical period of life that can decide the outcome in adult years. Participation in a variety of light intensity physical activities such as swimming is appropriate for any level of gross motor skill and often provides satisfaction and pleasure.

Angus Clarke, Ten years of the Cardiff Rett syndrome clinic, Cardiff University, UK.

The Cardiff Rett Syndrome clinic began with the support of Dr Alison Kerr from Glasgow more than ten years ago. The clinical team has included three consultants (clinical geneticist, paediatric neurologist and adult learning disability psychiatrist) and a physiotherapist. It is now also supported by an eye gaze technology representative, a support worker from Rett UK and sometimes by a music therapist. The clinic is not able to see patients either regularly or frequently as it can only be assembled occasionally (2-3 times per year): referrals are for one-off consultations. The referrals fall into four main categories: to address questions of diagnostic uncertainty, to discuss the questions that arise for families around the time of diagnosis, to consider care at the time of transition from paediatric to adult services and as a trouble-shooting assessment for difficult management problems.

He mentioned an example of the problems he was able to solve, but also mentioned that in many cases he was just helpless. He told us about the girl in her teens that was crying in pain for days. They did all the tests they could, examine all the potential issues and could not find any. Then he decided to try to stop topiramate medication for the seizures this girl presumably had. This made a change and the girl stopped crying.

Angus Clarke also stressed that the clinicians should not medicate the EEG of the Rett patient but seizures. Antiepileptic medication have numerous serious side effects and should be considered only in the case of a well-established diagnose of epilepsy.

 

Walter E Kaufmann, Clinical trials in Rett syndrome: opportunities and challenges.

DR Kaufmann has a very long experience with clinical trials, first with another rare disease called Fragile X syndrome. For Rett syndrome he was a principal investigator for the Phase I IGF1 (mecasermin) clinical trial that took place at Boston Children’s Hospital. Boston Children’s Hospital (BCH) is now recruiting subjects for the Phase 2 clinical trial entitled “Pharmacological Treatment of Rett Syndrome by Stimulation of Synaptic Maturation with IGF-1”.

http://www.rettsyndrome.org/research/clinical-trials

Challenges are many but the most important are scarcity of good outcome measures and biomarkers and regulatory challenges (regulatory agencies that are responsible for the marketing authorisation of the new medicinal products are FDA in the USA and EMA in Europe). Targeting regression period might be too late but developing a medicinal product for infants and small children and even the whole paediatric population is very challenging since the regulatory rules are very strict and also this population is more vulnerable.

He suggested some potential solutions. In his opinion the transition from animal models to human clinical trials should happen sooner than later and in younger population. Testing in younger population should produce better results and prove significant efficacy and benefits of the treatment more easily than in older population.

Trial design should consider heterogeneity and evolution in RETT.

Combining medicinal products with stimulation and teaching might be necessary for better results. Smart design, adaptive trials (An adaptive clinical trial is a clinical trial that evaluates a medical device or treatment by observing participant outcomes (and possibly other measures, such as side-effects) on a prescribed schedule, and modifying parameters of the trial protocol in accordance with those observations. The adaptation process generally continues throughout the trial, as prescribed in the trial protocol. Modifications may include dosage, sample size, drug undergoing trial, patient selection criteria… should be taken into account. Transferring knowledge from trials to clinic (outcome measures, biomarkers) might speed up the process. In clinical practise doctors should consider doing more pragmatic trials.

Huda Y. Zoghbi, Rett Syndrome: A winding path from clinic to bench and back to the clinic.

Her relationship with Rett syndrome goes back to 1983 when she met a beautiful 5 year-old girl with Rett syndrome at Texas Children’s hospital. She was a neurology resident at that time and Dr Alan Percy (child neurologist) was one of her mentors. A week later in the Blue Bird Circle Clinic she was seeing patients as resident. She chose to see a girl with the diagnosis of cerebral palsy, but at the moment this 12 year-old girl entered her office wringing her hands she knew that it was Rett syndrome she was dealing with. Seeing two girls with Rett in one week (when no U.S. physician had yet published on the syndrome) struck her as unusual and convinced her that there must be more. Digging into medical records brought to light many more cases. She met Dr Andreas Rett and watched him examine dozens of girls at the first U.S. conference in Baltimore. She was amazed how gentle, caring and curious he was. Dr Zoghbi was driven to pursue Rett through research and the fact that affected individuals were predominantly girls was a clue for her that the genetic defect was likely the cause of this disorder. Her search for the gene finally bore fruit in 1999 when the MECP2 gene was found in her laboratory by Ruthie Amir.

Now we know that the mutations in this same gene can also cause autism, bipolar disorder and juvenile onset schizophrenia. Through the use of the genetically-engineered mice, the Zoghbi lab learned that the brain is sensitive to the levels of MeCP2 protein and that both decreases and increases in MeCP2 levels (or function) can lead to neurological and behavioural features that are also observed in humans. They showed that the protein is needed throughout life and that normalizing its levels can reverse disease-like features in a mouse model of the human MECP2 duplication syndrome. Zoghbi and collaborators have more recently uncovered circuit abnormalities in both the Rett and duplication mouse models.

Huda Zoghbi stressed the complexity of the MECP2 function. There are around 2500 genes affected by it which are either up or down regulated. 50% of these genes are causing some kind of intellectual disability. Part of the brain responsible for the cognitive functions is the most sensitive one and can be easily interrupted causing different memory and behavioural issues.

Inhibitory neurons are critical for proper brain function, and their dysfunction is implicated in several disorders, including autism, schizophrenia, and Rett syndrome. These neurons are heterogeneous, and it is unclear which subtypes contribute to specific neurological phenotypes. Deletion of the mecp2 mouse homolog, from the two most populous subtypes, parvalbumin-positive (PV+) and somatostatin-positive (SOM+) neurons partially impairs the affected neurons, allowing to assess the function of each subtype without profound disruption of neuronal circuitry. Mice lacking MeCP2 in either PV+ or SOM+ neurons have distinct, non-overlapping neurological features: mice lacking MeCP2 in PV+ neurons developed motor, sensory, memory, and social deficits, whereas those lacking MeCP2 in SOM+ neurons exhibited seizures and stereotypies. The findings indicated that PV+ and SOM+ neurons contribute to complementary aspects of the Rett phenotype and may have modular roles in regulating specific behaviours.

http://www.cell.com/neuron/abstract/S0896-6273%2815%2900923-X

Deep brain stimulation (DBS) is a neurosurgical procedure introduced in 1987 involving the implantation of a medical device called a neurostimulator (sometimes referred to as a ‘brain pacemaker’), which sends electrical impulses, through implanted electrodes, to specific targets in the brain (brain nuclei). Dr Zoghbi and her team showed that deep brain stimulation of a specific neural network in the fornix improved learning and memory in a Rett syndrome mouse model. 200K implants are already used in humans for the treatment of tremors and dystonia in Parkinson disease. In her experiment mice had a DBS treatment for 1h/day. Huda stressed the importance of training at the same time with DBS in order for the procedure to be more effective. Also the timing should be as early as possible. Next step is to plan a clinical trial with DBS.

Recently Huda Zoghbi has been working on Antisense oligonucleotide (ASO) therapy for MECP2 duplication syndrome.

Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene “off”. This is because mRNA has to be single stranded for it to be translated. More details about this research on:

https://reverserett.org/research/project/investigating-the-potential-of-antisense-oligonucleotide-therapy-for-mecp2-duplication-syndrome/

Free Communications sessions were chaired by Franco Laccone from the Medical University of Vienna.

Development and characterization of a human Rett syndrome cell model using a non-integrating reprogramming strategy.

The purpose of this study was to establish a human cell model for Rett syndrome via a non-integrating direct reprogramming method, to accurately characterize it and to evaluate in vitro protein replacement therapy using a TAT-MeCP2 fusion protein.

This type of protein replacement therapy was a subject of the winning poster by Anna Huber from the Medical University of Vienna. The full title is: The blood-brain barrier as an important factor for drug delivery of “Rett-Therapeutics” to the brain.

TAT (TransActivator of Transcription) peptide is encoded by TAT gene in HIV-1 and has cell-penetrating abilities.

Drug delivery to the central nervous system (CNS) remains a major pharmaceutical challenge due to the non-permeability of blood-brain barrier (BBB). In order to deliver the functional MeCP2 into the CNS it has to overcome the BBB, which could be enhanced by the modification of the protein with a cell penetration improving strategy such as the addition of a TAT sequence. An in vitro model of the mouse BBB was successfully established and characterized. Transport data with this model revealed a distinct barrier by brain endothelial cell layers for the transport of the investigated TAT-MeCP2 proteins, but also showed that significant amounts of TAT-protein preparations were able to cross the BBB in-vitro.

Brain SRB1 modulation as a possible player in Rett syndrome pathogenesis, Giuseppe Valacchi.

http://www.cellbiolabs.com/sites/default/files/STA-630-srb1-elisa-kit.pdf

Recent data highlight that lipid metabolism is not normal in brain and liver of MECP2-null mice. In addition, altered plasma lipid profile in RTT patients has been observed. Furthermore redox modulation of the HDL receptor SRB1 was observed in skin fibroblasts isolated from patients with Rett syndrome. Cholesterol within high-density lipoprotein (HDL) is removed from circulation through binding and endocytosis of the Scavenger Receptor Class B Member 1 (SRB1).

The results of the study showed that SRB1 expression decreases significantly during the progression of the disease in MECP2-null mice and almost disappears in 17 weeks old MECP2-null mice. They were able to show that SRB1 co-localized with tubulin III, suggesting its presence in the neurons. These results demonstrate that SRB1 which is one of the main proteins belonging to cholesterol regulatory network is altered in RTT animal model thus providing the proof of principle that cholesterol metabolism may be taken into account as a new target for the treatment of specific features of RTT pathology.

https://clinicaltrials.gov/ct2/show/NCT02563860?term=rett&rank=28

Rett Syndrome Europe General Assembly Meeting.

RSE board members and present members gathered on the Saturday late afternoon session for the annual General assembly meeting where Danijela Szili was elected for another term. Gillian Townend and other communication specialists of the Maastricht Rett Centre were also present at the beginning of the GAM as external observers. G. Townend presented a draft of a questionnaire written in English language and aimed at families in order to populate data for the preparation of communication guidelines by the Maastricht Rett Centre. Members of RSE present that day voluntarily proposed to make a translation of the questionnaire in their native language.

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