MECP2 Mutations Research Articles

Deep brain stimulation for Rett syndrome

Mutations in the gene MECP2 cause an intellectual-disability disorder called Rett syndrome. In a mouse model, electrical stimulation of deep brain regions is found to ameliorate some of the features of the syndrome. See Letter p.430 Rett syndrome is a genetic disorder that causes profound intellectual disability and other impairments. Huda Zoghbi and colleagues now show that in a mouse model of the disorder, a two-week course of daily deep-brain stimulation of the fimbria-fornix — part of the brain that provides input to the hippocampus — restored hippocampal-dependent memory when tested three weeks after the end of the treatment. It also restored hippocampal long-term potentiation and neurogenesis. These findings indicate that deep-brain stimulation, which is already used in the treatment of motor diseases such as Parkinson's disease and dystonia, could be a viable approach to mitigating cognitive impairment in Rett syndrome and other disorders of childhood intellectual disability.

Visual evoked potentials detect cortical processing deficits in Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation of the X-linked MECP2 gene and characterized by developmental regression during the first few years of life. The objective of this study was to investigate if the visual evoked potential (VEP) could be used as an unbiased, quantitative biomarker to monitor brain function in RTT. We recorded pattern-reversal VEPs in Mecp2 heterozygous female mice and 34 girls with RTT. The amplitudes and latencies of VEP waveform components were quantified, and were related to disease stage, clinical severity, and MECP2 mutation type in patients. Visual acuity was also assessed in both mice and patients by modulating the spatial frequency of the stimuli. Mecp2 heterozygous female mice and RTT patients exhibited a similar decrease in VEP amplitude that was most striking in the later stages of the disorder. RTT patients also displayed a slower recovery from the principal peak of the VEP response that was impacted by MECP2 mutation type. When the spatial frequency of the stimulus was increased, both patients and mice displayed a deficit in discriminating smaller patterns, indicating lower visual spatial acuity in RTT. VEP is a method that can be used to assess brain function across species and in children with severe disabilities like RTT. Our findings support the introduction of standardized VEP analysis in clinical and research settings to probe the neurobiological mechanism underlying functional impairment and to longitudinally monitor progression of the disorder and response to treatment.

Gene Therapy for Rett Syndrome: Prospects and Challenges

Rett syndrome (RTT) is a neurological disorder that affects females and is caused by loss-of-function mutations in the X-linked gene MECP2. Deletion of Mecp2 in mice results in a constellation of neurological features that resemble those seen in RTT patients. Experiments in mice have demonstrated that restoration of MeCP2, even at adult stages, reverses several aspects of the RTT-like pathology suggesting that the disorder may be inherently treatable. This has provided an impetus to explore several therapeutic approaches targeting RTT at the level of the gene, including gene therapy, activation of MECP2 on the inactive X chromosome and read-through and repair of RTT-causing mutations. Here, we review these different strategies and the challenges of gene-based approaches in RTT.

Genotype-phenotype relationship among Egyptian children with Rett syndrome.

Rett syndrome (RTT) is an X-linked dominant neurodegenerative disorder with various MECP2 mutations. RTT is one of the most common causes of severe intellectual and complex disability in girls. Therefore, the aims of the study were as follows: to highlight the clinical manifestations of RTT; to present the genotype-phenotype relationship; and to assess the possible relation between severity score, clinical manifestations, and MECP2 gene mutations. The present cross-sectional study included 15 girls with typical RTT, diagnosed according to the international criteria of RTT. All included patients were followed up at the pediatric neurology clinic, Cairo University Specialized Pediatric Hospital. They were subjected to screening of the entire coding region of the MECP2 gene (MECP2A and MECP2B) using denaturing high-performance liquid chromatography. The clinical severity was assessed among RTT cases using the International Scoring System. Stereotypic hand movements were present in all cases, acquired microcephaly was present in 73.3% of cases, autistic features in 66.7% of cases, recurrent seizures in 53.3% of cases, delayed language development in 46.6% of cases, deterioration of speech in 53.3% of cases, and growth retardation and peripheral vasomotor changes in 46.6% of cases. Positive mutations were detected in 10 cases (66.66%): heterozygous for p.R270X mutation (three cases), heterozygous for p.R255X mutation (three cases), and heterozygous for p.R168X nonsense mutation (four cases). Microcephaly, seizures, growth retardation, and autistic features were more frequent in patients with a mutated gene; it was also observed that walking ability was more frequent in patients without a mutation.; thus, genotype-phenotype relationship was confirmed. The relationship between severity score and MECP2 mutation was detected in three cases with severe RTT, but there was no relationship between the severity score and specific MECP2 mutation. There was a relationship between the severity score and the clinical manifestations of RTT. Mutations of MECP2 analysis were detected in 66.7% of RTT cases. There were relationships between the severity score, clinical manifestations, and MECP2 gene mutations. However, there was no relationship between the severity score and specific MECP2 gene mutation.

Karyopherin α 3 and Karyopherin α 4 Proteins Mediate the Nuclear Import of Methyl-CpG Binding Protein 2

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein with important roles in regulating chromatin structure and gene expression, and mutations in MECP2 cause Rett syndrome (RTT). Within the MeCP2 protein sequence, the nuclear localization signal (NLS) is reported to reside between amino acids 255-271, and certain RTT-causing mutations overlap with the MeCP2 NLS, suggesting that they may alter nuclear localization. One such mutation, R270X, is predicted to interfere with the localization of MeCP2, but recent in vivo studies have demonstrated that this mutant remains entirely nuclear. To clarify the mechanism of MeCP2 nuclear import, we isolated proteins that interact with the NLS and identified karyopherin α 3 (KPNA3 or Kap-α3) and karyopherin α 4 (KPNA4 or Kap-α4) as key binding partners of MeCP2. MeCP2-R270X did not interact with KPNA4, consistent with a requirement for an intact NLS in this interaction. However, this mutant retains binding to KPNA3, accounting for the normal localization of MeCP2-R270X to the nucleus. These data provide a mechanism for MeCP2 nuclear import and have implications for the design of therapeutics aimed at modulating the function of MeCP2 in RTT patients.

MiR-199a Links MeCP2 with mTOR Signaling and Its Dysregulation Leads to Rett Syndrome Phenotypes.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR), which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling.

Is it possible to diagnose Rett syndrome before classical symptoms become obvious? Review of 24 Danish cases born between 2003 and 2012.

Rett syndrome (RTT) is a neurodevelopmental disorder that affects mainly females; it results in multiple disabilities and carries a risk of medical comorbidities. Early diagnosis is important to help establish the best treatment opportunities and preventive care in order to slow down the progression of symptoms. We wanted to test our hypothesis that it is possible to diagnose RTT before the classical symptoms become obvious. We analysed development and symptoms before and at the time of the RTT diagnosis, as well as the symptoms that triggered MECP2 mutation analysis, in a cohort of girls with RTT born in Denmark between 2003 and 2012. Twenty-four girls were included, and 87.5% of these girls were diagnosed when the classical RTT symptoms were recognized. However, parents were concerned about their daughters between 3 and 58 months prior to the RTT diagnosis, and they felt that the professionals did not share their concern in the beginning. When reviewing medical files and questionnaires, we noted that the majority of girls did have combinations of concerning symptoms such as developmental delay and a collection of subtle signs such as autistic traits, placidity, floppiness with suspicion of muscular or mitochondrial diseases, hair pulling, teeth grinding, development of incontinence and problems with initiating movements. We conclude that many individuals with MECP2 mutation exhibit characteristics that should raise suspicion for RTT, prior to evolution of the core clinical criteria. As RTT is a rare disease, it is of importance to constantly educate clinicians for heightened awareness of RTT.

Disturbance of cardiac gene expression and cardiomyocyte structure predisposes Mecp2-null mice to arrhythmias.

Methyl-CpG-binding protein 2 (MeCP2) is an epigenetic regulator of gene expression that is essential for normal brain development. Mutations in MeCP2 lead to disrupted neuronal function and can cause Rett syndrome (RTT), a neurodevelopmental disorder. Previous studies reported cardiac dysfunction, including arrhythmias in both RTT patients and animal models of RTT. In addition, recent studies indicate that MeCP2 may be involved in cardiac development and dysfunction, but its role in the developing and adult heart remains unknown. In this study, we found that Mecp2-null ESCs could differentiate into cardiomyocytes, but the development and further differentiation of cardiovascular progenitors were significantly affected in MeCP2 deficiency. In addition, we revealed that loss of MeCP2 led to dysregulation of endogenous cardiac genes and myocardial structural alterations, although Mecp2-null mice did not exhibit obvious cardiac functional abnormalities. Furthermore, we detected methylation of the CpG islands in the Tbx5 locus, and showed that MeCP2 could target these sequences. Taken together, these results suggest that MeCP2 is an important regulator of the gene-expression program responsible for maintaining normal cardiac development and cardiomyocyte structure.

Applying the ethoexperimental approach to neurodevelopmental syndrome research reveals exaggerated defensive behavior in Mecp2 mutant mice

Rett syndrome is a Pervasive Developmental Disorder (PDD) associated with de novo mutations of the methyl CpG-binding protein 2 (MECP2) gene. Mecp2 functions as a transcription factor that regulates the expression of hundreds of genes. Identification of the role of Mecp2 in specific neurodevelopmental symptoms remains an important research aim. We previously demonstrated that male mice possessing a truncation mutation in Mecp2 are hyper-social. We predicted that reduced fear or anxiety might underlie this enhanced affiliation. In order to probe risk assessment and anxiety-like behavior, we compared Mecp2 truncation mutants to their wild-type littermates in the elevated plus maze and elevated zero maze. Additionally, subjects were administered the mouse defense test battery to evaluate unconditioned fear- and panic-like behavior to a graded set of threat scenarios and a predator stimulus. Mutant mice showed no significant changes in anxiety-like behavior. Yet, they displayed hyper-reactive escape and defensive behaviors to an animate predatory threat stimulus. Notably, mutant mice engaged in exaggerated active defense responding to threat stimuli at nearly all phases of the fear battery. These results reveal abnormalities in emotion regulation in Mecp2 mutants particularly in response to ecologically relevant threats. This hyper-responsivity suggests that transcriptional targets of Mecp2 are critical to emotion regulation. Moreover, we suggest that detailed analysis of defensive behavior and aggression with ethologically relevant tasks provides an avenue to interrogate gene-behavior mechanisms of neurodevelopmental and other psychiatric conditions.

MeCP2 in the enteric nervous system.

Rett syndrome (RTT) is an intellectual deficit and movement disorder that develops during early childhood in girls. Affected children are normal until 6-18 months of age, after which symptoms begin to appear. Most cases of RTT are due to mutations in the MeCP2 gene leading to disruption of neuronal communication in the central nervous system. In addition, RTT patients show peripheral ailments such as gastrointestinal (GI), respiratory, and cardiac dysfunction. The etiology of intestinal dysfunction in RTT is not well-understood. Reports on the presence of MeCP2 in the peripheral nervous system are scant. As such we examined the levels of MeCP2 in human and murine GI tissue and assessed MeCP2 expression at various developmental stages. Immunohistochemistry for MeCP2, HuC/D, juvenile beta tubulin, and GFAP was performed on human and murine intestine. Western blots of these same tissues were probed with MeCP2, vAChT, nNOS, and beta-actin antibodies. MeCP2 is expressed throughout the GI tract. MeCP2 is expressed specifically in the enteric nervous system of the GI tract. MeCP2 is expressed in the GI tract throughout development with appearance beginning at or before E11.5 in the murine intestine. The proof of MeCP2 expression in enteric neurons suggests that the GI dysmotility in Rett may arise from enteric network dysfunction secondary to MeCP2 mutation.

Differentiation of multipotent neural stem cells derived from Rett syndrome patients is biased toward the astrocytic lineage.

BackgroundRett syndrome (RTT) is one of the most prevalent neurodevelopmental disorders in females, caused by de novo mutations in the X-linked methyl CpG-binding protein 2 gene, MECP2. Although abnormal regulation of neuronal genes due to mutant MeCP2 is thought to induce autistic behavior and impaired development in RTT patients, precise cellular mechanisms underlying the aberrant neural progression remain unclear.ResultsTwo sets of isogenic pairs of either wild-type or mutant MECP2-expressing human induced pluripotent stem cell (hiPSC) lines were generated from a single pair of 10-year-old RTT-monozygotic (MZ) female twins. Mutant MeCP2-expressing hiPSC lines did not express detectable MeCP2 protein during any stage of differentiation. The lack of MeCP2 reflected altered gene expression patterns in differentiated neural cells rather than in undifferentiated hiPSCs, as assessed by microarray analysis. Furthermore, MeCP2 deficiency in the neural cell lineage increased astrocyte-specific differentiation from multipotent neural stem cells. Additionally, chromatin immunoprecipitation (ChIP) and bisulfite sequencing assays indicated that anomalous glial fibrillary acidic protein gene (GFAP) expression in the MeCP2-negative, differentiated neural cells resulted from the absence of MeCP2 binding to the GFAP gene.ConclusionsAn isogenic RTT-hiPSC model demonstrated that MeCP2 participates in the differentiation of neural cells. Moreover, MeCP2 deficiency triggers perturbation of astrocytic gene expression, yielding accelerated astrocyte formation from RTT-hiPSC-derived neural stem cells. These findings are likely to shed new light on astrocytic abnormalities in RTT, and suggest that astrocytes, which are required for neuronal homeostasis and function, might be a new target of RTT therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0121-2) contains supplementary material, which is available to authorized users.

P192 – 2531: Overview of Rett syndrome associated with MECP2 mutation: Analysis of 7 patients

Objective The aim of the present study was to report our experience with Rett syndrome (RTT) associated with MECP2 mutation. Methods Seven consecutive patients diagnosed with RTT associated with MECP2 mutation in our child neurology clinic were analyzed in terms of clinical findings retrospectively. Diagnosis were compared according to revised diagnostic criteria (2010) for RTT. Results There were 7 girls, age range, 2.3 to 10.2 years. Age at the time of diagnosis were 2 to 5 years. Although 2 of the child had history of preterm birth. All of the patient had normal psychomotor development in first six months of life. Therefore none of the patient had any exclusion criteria. All of the patients had neuromotor regression period. However only 3 of patients followed by recovery or stabilization. All of the patients had complete loss of acquired purposeful hand skills, stereotypic hand movements and partial or complete loss of acquired spoken language. One of the patients had never experienced independent walking. Independent walking had been possible in three patients. Developmental regression was seen in all the patients, and the average onset of age was 19 months. Although all of patients had epileptic abnormality on EEG, only 2 patients manifested with epilepsy that required antiepileptic medication. When we examine patients for supportive criteria; we observed breathing disturbances when awake in 3, bruxism in 4, impaired sleep pattern in 2, peripheral vasomotor disturbances in 4, scoliosis in 3, growth retardation 6, small cold hands and feet in 7, inappropriate laughing/screaming spells in 5, diminished response to pain in 7, intense eye communication in 2 cases. All of the patients diagnosed as typical RTT according to 2010 revised diagnostic criteria. Conclusion This study supported that revised diagnostic criteria (2010) for RTT is a useful tool for clinicians for diagnosis of RTT.

OP22 – 2962: Aging in females with Rett syndrome. Follow up study of Hungarian patients with Rett syndrome

Objective Rett syndrome is a neurological disease affecting mainly females. The arrest of brain development is caused mainly by the X-linked, MECP2 mutation. There are only few and individual case reports about living 50–70-year-old women with Rett syndrome, therefore little is known about the aging process with this syndrome. Methods After publishing the causative role of MECP2 mutation in Rett syndrome in 1999, at the Department of Paediatrics, University of Pecs we started to examine all Hungarian patients with suspected Rett syndrome in collaboration with our Medical Genetics Institute. 88 children with clinically suspected Rett syndrome were admitted to our ward between 2001 and 2002. The youngest patient was 3 years old, the oldest one 21 years old. Very detailed history taking, neurological, orthopaedic, genetic, EEG and psychological examinations were administered with every child and their families. All patients were video-recorded. MECP2 mutation was found in 35 girls. Results 26 females entered adulthood till this time (their age is over 18 years). They were re-evaluated. Only the patients with proven MECP2 mutation were followed. Epileptic, orthopaedic and mental status, gait ability, growth, nutritional and gastrointestinal status, breathing patterns, hand or other stereotypes, sleeping patterns, behaviour were checked. In our study we give a video-presentation about the characteristic changes of the health status of females with Rett syndrome from the very early age to their twenties-thirties. Conclusion Good life circumstances and available medical care seems to ensure better life expectancy to the females with Rett syndrome. The increased longevity of the patients requires a specific and individually tailored care. The families of females with Rett syndrome and the societies have to prepare themselves to support adults with childhood illnesses optimally.

P193 – 2860: Many faces of Rett syndrome: Is there still a diagnostic delay?

Objective Rett syndrome is the second most common neurodevelopmental genetic disorder in girls. Major clinical features include developmental regression after a normal period of development, autistic behavior, loss of purposeful hand use, stereotypical hand movements, abnormal breathing pattern, peripheral vasomotor disturbances and seizures. MECP2 gene mutations can be detected in up to 90–95% of patients with classical Rett syndrome. Methods In order to document the clinical course in this syndrome, we have included 15 consecutive patients in whom MECP2 mutations were detected. Results Age range was 3–22 years (median 15 years). First admission to a physician with a suspicion of abnormal development was between 8 months to 4 years. Diagnostic delay on the bedside was calculated as median 1 year (0–3.5 years). Stereotypical hand movements developed between 8 months – 5 years (median 2 years). One patient did not have normal early developmental milestones. Loss of purposeful hand movements was seen between 1–9 years (median 2 years). Of note, in one patient loss occurred at 9 years of age and in another 3-year-old, purposeful hand use was never observed. Microcephaly was detected in 12 patients. During a mean course of 5.7 years (2 months to 18 years), two of the patients lost independent ambulation (6 and 10 years). Other than stereotypical hand movements 5 patients had additional stereotypies including pelvic tilt, truncal rocking motion and spitting behavior. Conclusion Despite recent availability of MECP2 testing, there is still a considerable delay in diagnosis, due to clinical variability in terms of appearance of core symptoms and course.

P189 – 2279: Is the regression period in Rett syndrome well defined and easy to recognize?

Objective In order to increase knowledge of regression as a clue to diagnose Rett syndrome (RTT), we want to investigate if the regression period (RP) is well defined and easy to recognize. Methods Medical records (including questionnaires and interviews) were reviewed concerning the RP in children with clinical Rett syndrome and a MECP2 mutation referred to the nation-wide Centre for Rett syndrome in Denmark in the years 2012–2014. Results Ten girls with RTT (8 typical/2 atypical) and a MECP2 mutation aged 1.7–5.3 years (mean 3.3 years; median 2.8 years) were referred to the centre and evaluated within 13–152 days (mean 58.3 days; median 41.5 days). Seven of the girls were post regression at this first evaluation. In the other 3 cases it was difficult to conclude but time showed that 2 girls were still in regression at the first visit. The girls were in RP in the range of about 4–35 months (mean 14.8 mo; median 12 mo). Before the RTT diagnosis 5 of the girls were referred to psychiatric evaluation at a time that turned out to be in the RP. One of these girls and 3 others had a muscular biopsy before (2), under (1) or after (1) an unrecognized RP. One girl had severe symptoms of epilepsy and adverse reactions to medication in RP. In 6 cases the RP along with other symptoms as handstereotypies led to suspicion of RTT. Five of the girls were diagnosed in the RP and the other 5 after the RP. Conclusion In most of our cases, RP was not well defined or easily recognized. It might be overlooked because of its long and slow progression as well as co-morbidities like hypotonia or epilepsy. We conclude that more information regarding regression in RTT is needed to facilitate early diagnosis, counselling and treatment.

P191 – 2505: Two siblings with a CDKL5 mutation: Genotype and phenotype evaluation

Objective This is the second report of a family with a recurrence of a CDKL5 mutation (c. 283-3_290del) in two sisters. Their phenotype is different despite an identical genotype Clinically CDKL5 patients resemble those with Rett syndrome, caused by a MECP2 mutation, who experience a regression, after an initial normal development. However, CDKL5 patients experience a developmental delay from from birth onwards and seizures starts the first three months of life. Methods Advanced genetic evaluations of the sisters and parents, the electropherogram, clinical and electroencephalographic (EEG) features of both sisters are discussed. Results In the youngest patient, who was known to us prior to appearance of the clinical symptoms, neurological and EEG evaluations at the age of 6 weeks were normal. Infantile spasms evolved at the age of 10 weeks. Simultaneously, she showed a psychomotor regression and deterioration of the EEG into a severe epileptic pattern. Her older sister, was from the start severely hypotonic, had a developmental delay (without regression) and feeding problems and experiences seizures from the age of 8 weeks. In both seizures are refractory. The phenotype of the youngest sister is worse than that of her older sister, despite an identical genotype. This may be explained by differences in X-chromosomal inactivation and other unknown epigenetic and environmental factors. Unfortunately, X inactivation pattern could not be determined in the youngest sister. Both parents tested negative for the mutation in all tissues, but germline mosaicism is likely. Conclusion This report of familial recurrence with suspected germline mosaicism in a healthy parent, has important consequences for genetic counselling. Although it is not possible to predict an exact recurrence risk, it is likely to be increased.

OP21 – 2277: Centre for Rett syndrome in Denmark – Clinical follow-up with short distance to research

Objective Since 2011 persons with Rett syndrome (RTT) in Denmark have had the possibility for life-long multi-disciplinary follow-up at the nation-wide Centre for Rett syndrome. In 2007–2011 a PhD study was performed. We want to describe experiences from the centre focused on clinical follow-up, counselling and inclusion in research projects. Methods Retrospective survey (31 December 2014); parameters: age of patients, mutations, outpatient visits, local counselling visits, participation in research projects. Only alive patients were included and all ages were calculated by 31 December 2014. Results 92 patients (86% of registered Danish patients) with a clinical RTT diagnosis visited the centre 2007–2014. 64 of these patients were evaluated 2012–2014; mean of 2.5 visits; total 160; range 1–7; ages 2.5–58.4 years; mean 19.8 years; median 17.0 years. 60 MECP2 mutations, one CDKL5, 3 without mutation. Of the 92 patients, 28 were only seen in 2007–2011: aged 12.4 to 60.3 years; mean 29.8 years; median 30.6 years. 47 were re-evaluated in 2012–2014: aged 4.6 to 58.4 years; mean 22.2 years; median 18.26 years. Fifty-seven counselling visits were performed at schools, day-care centres and homes for disabled persons. Sixty-one (58 from the alive cohort) patients were included in the PhD study “Bone mass and fracture occurrence in Danish patients with Rett syndrome” (Gitte Roende) 2007–2011. In 2013–2014, 42 persons (3–45 years) participated in pilot studies regarding physiotherapeutic outcome measures. Conclusion A large proportion of persons with RTT are referred to the centre; however adults to a lesser degree. Once assessed by the multi-disciplinary team, they usually choose to continue regular outpatient visits and counselling. There are good opportunities for implementation of research projects (prospective studies and follow-up of clinical data) and many show interest in participation. In 2015 they will be invited to the PhD study “Living an active life with Rett syndrome”.

P198 – 3022: GABAergic dysfunction in neurons derived from induced pluripotent stem cells of patients with Rett syndrome

Objective Induced pluripotent stem cells (iPs cells) are a promising tool for the study of neurological diseases. Disruption of GABAergic activity in Rett Syndrome (RTT) can be related, at least in part, with a failure to complete the GABA functional switch that occurs during normal development, from excitation in immature neurons to inhibition in mature neurons. This switch is mainly mediated by a developmental increase in KCC2 expression, a co-transporter that drives Cl-efflux outside neurons, with the consequent decrease in intracellular Cl-concentration and GABA-mediated hyperpolarization in mature neurons. We documented reduced KCC2 levels in the cerebrospinal fluid of RTT patients and aimed to test GABAergic function in neurons derived from RTT patients' iPs cells. Methods Human skin biopsies were collected in accordance with European and National ethical regulation and IPS cells were generated from fibroblast cells upon infection with a retroviral vector expressing four transcription factors (Oct4, Sox2, Klf4, and Myc). IPs cell lines were obtained from patients carrying MECP2 mutations (R306C and R255X) and controls. Neuronal cortical populations were derived in a monolayer culture system using the dual-SMAD inhibition protocol. Perforated patch recordings were performed in these neurons and GABA-evoked postsynaptic currents were measured to evaluate GABA A receptor equilibrium potencial. Adittionally, we performed molecular studies in a RTT mouse model (Mecp2tm1.1Bird/J). Results Preliminary data indicate that recordings from RTT cells exhibit a GABA A receptor equilibrium potential that is more positive than in recordings from control cells, indicating changes in chloride gradient, characteristic of an immature state. The level of KCC2 protein expression is lower in Mecp2-KO mice when compared to control littermates, as addressed by western blot analysis of 6 week old hippocampi. Conclusion Results form the animal model, iPs cell-based models and cerebrospinal fluid studies suggest a GABAergic dysfunction in RTT, including alterations in KCC2 expression and/or activity.

Mutations in epilepsy and intellectual disability genes in patients with features of Rett syndrome

Rett syndrome and neurodevelopmental disorders with features overlapping this syndrome frequently remain unexplained in patients without clinically identified MECP2 mutations. We recruited a cohort of 11 patients with features of Rett syndrome and negative initial clinical testing for mutations in MECP2. We analyzed their phenotypes to determine whether patients met formal criteria for Rett syndrome, reviewed repeat clinical genetic testing, and performed exome sequencing of the probands. Using 2010 diagnostic criteria, three patients had classical Rett syndrome, including two for whom repeat MECP2 gene testing had identified mutations. In a patient with neonatal onset epilepsy with atypical Rett syndrome, we identified a frameshift deletion in STXBP1. Among seven patients with features of Rett syndrome not fulfilling formal diagnostic criteria, four had suspected pathogenic mutations, one each in MECP2, FOXG1, SCN8A, and IQSEC2. MECP2 mutations are highly correlated with classical Rett syndrome. Genes associated with atypical Rett syndrome, epilepsy, or intellectual disability should be considered in patients with features overlapping with Rett syndrome and negative MECP2 testing. While most of the identified mutations were apparently de novo, the SCN8A variant was inherited from an unaffected parent mosaic for the mutation, which is important to note for counseling regarding recurrence risks.

The methyl-CpG-binding domain (MBD) is crucial for MeCP2's dysfunction-induced defects in adult newborn neurons.

Mutations in the human X-linked gene MECP2 are responsible for most Rett syndrome (RTT) cases, predominantly within its methyl-CpG-binding domain (MBD). To examine the role of MBD in the pathogenesis of RTT, we generated two MeCP2 mutant constructs, one with a deletion of MBD (MeCP2-ΔMBD), another mimicking a mutation of threonine 158 within the MBD (MeCP2-T158M) found in RTT patients. MeCP2 knockdown resulted in a decrease in total dendrite length, branching, synapse number, as well as altered spontaneous Ca2+ oscillations in vitro, which could be reversed by expression of full length human MeCP2 (hMeCP2-FL). However, the expression of hMeCP2-ΔMBD in MeCP2-silenced neurons did not rescue the changes in neuronal morphology and spontaneous Ca2+ oscillations, while expression of hMeCP2-T158M in these neurons could only rescue the decrease in dendrite length and branch number. In vivo over expression of hMeCP2-FL but not hMeCP2-ΔMBD in adult newborn neurons of the dentate gyrus also rescued the cell autonomous effect caused by MeCP2 deficiency in dendrites length and branching. Our results demonstrate that an intact and functional MBD is crucial for MeCP2 functions in cultured hippocampal neurons and adult newborn neurons.