MECP2 Mutations Research Articles

A novel mutation in the X‐linked cyclin‐dependent kinase‐like 5 (CDKL5) gene associated with a severe Rett phenotype

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have recently been reported in patients with severe neurodevelopmental disorder characterized by early-onset seizures, infantile spasms, severe psychomotor impairment and very recently, in patients with Rett syndrome (RTT)-like phenotype. Although the involvement of CDKL5 in specific biological pathways and its neurodevelopmental role have not been completely elucidated, the CDKL5 appears to be physiologically related to the MECP2 gene. Here we report on the clinical and CDKL5 molecular investigation in a very unusual RTT case, with severe, early-neurological involvement in which we have shown in a previous report, a novel P388S MECP2 mutation [Conforti et al. (2003); Am J Med Genet A 117A: 184-187]. The patient has had severe psychomotor delay since the first month of life and infantile spasms since age 5 months. Moreover, at age 5 years the patient suddenly presented with renal failure. The severe pattern of symptoms in our patient, similar to a CDKL5 phenotype, prompted us to perform an analysis of the CDKL5, which revealed a novel missense mutation never previously described. The X-inactivation assay was non-informative. In conclusion, this report reinforces the observation that the CDKL5 phenotype overlaps with RTT and that CDKL5 analysis is recommended in patients with a seizure disorder commencing during the first months of life.

InterRett, a model for international data collection in a rare genetic disorder

Rett syndrome (RTT) is a rare genetic disorder within the autistic spectrum. This study compared socio-demographic, clinical and genetic characteristics of the international database, InterRett, and the population-based Australian Rett syndrome database (ARSD). It also explored the strengths and limitations of InterRett in comparison with other studies. A literature review compared InterRett with RTT population-based and case-based studies of 30 or more cases that investigated genotype and/or phenotype relationships. Questionnaire data were used to determine case status and to investigate the comparability of InterRett and ARSD. Twenty-four case series, five population-based studies and a MECP2 mutation database were identified of which 21 (70%) collected phenotype and genotype data. Only three studies were representative of their underlying case population and many had low numbers. Of 1114 InterRett subjects, 935 born after 1976 could be verified as Rett cases and compared with the 295 ARSD subjects. Although more InterRett families had higher education and occupation levels and their children were marginally less severe, the distribution of MECP2 mutation types was similar. The InterRett can be used with confidence to investigate genotype phenotype associations and clinical variation in RTT and provides an exemplary international model for other rare disorders.

A Temporal Threshold for Formaldehyde Crosslinking and Fixation

BackgroundFormaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined.MethodologyUsing a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells. The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process. We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking, also fails to capture these transient interactions.Conclusions/SignificanceOur findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research.

Methyl-CpG-binding protein 2 gene and CDKL5 gene mutation in patients with Rett syndrome: analysis of 177 Chinese pediatric patients

To study the spectrum of mutations in methyl-CpG-binding protein 2 gene (MECP2) and cyclin-dependent kinase-like 5 gene (CDKL5) in Chinese pediatric patients with Rett syndrome (RTT), and establish a simple, quick, and efficient gene test method as well as screen a strategy of genetic diagnosis for RTT. Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes of 117 pediatric patients diagnosed from 1987 to 2007. PCR was used to amplify the exons 1 - 4 of MECP2 using published primers. If no mutation was identified after screening exons 2 - 4, exon 1 was screened. If no mutation was identified in MECP2 by sequencing, multiplex ligation dependent probe amplification (MLPA) was employed to screen for large deletions by using P015C kit. If no mutation was identified in the MECP2 by sequencing and MLPA respectively, then the coding region of CDKL5 was screened by denaturing high performance liquid chromatography (DHPLC). The total mutation frequency in MECP2 and CDKL5 genes among all RTT patients was 82%. MECP2 mutations were found in 86% (137/159) of the patients with classical RTT and in 44% (8/18) of those with atypical RTT. Most of the mutations were missense mutations, accounting for 39%, followed in order of frequency by nonsense mutations 28%, frame shift mutations 17% and large deletions 14.5%. The eight most frequent MECP2 mutations were p.T158M (13%), p.R168X (12%), c.806delG (7%), p.R255X (6%), p.R270X (5%), p.R133C (5%), p.R306C (4%), and p.R106W (3%), with p.T158M as the most common of the MECP2 mutations and c.806delG as a hotspot mutation in Chinese patients with RTT. Only one synonymous mutation was identified in CDKL5. The spectrum of MECP2 mutations within the mainland Chinese RTT patients is similar to that of those patients reported in the world. p.T158M, p.R168X, c.806delG, p.R255X, p.R270X, p.R133C, p.R306C, and p.R106W are the hotspot mutations of MECP2 and c.806delG is a specific hotspot mutation in Chinese patients with RTT. The most effective method to screen mutations is to screen the exon 4. MLPA is an effective supplement to the routine methods.

Rett syndrome and long‐term disorder profile

In a cohort of 103 females clinically diagnosed with Rett syndrome (RTT), 91 had a detectable MECP2 mutation. Emphasis on details of natural history facilitated grouping of females with the same MECP2 mutation and the development of so-called disorder profiles. Some examples of disorder profiles of different recurrent MECP2 mutations are discussed. RTT females with the frequently recurrent R133C and R306C missense mutations and those with intragenic deletions in the C-terminus of MECP2 deserve more attention in larger studies as their development is different and milder in the long term. RTT females with the T158M missense mutation are often atypical with mainly behavioral characteristics in infancy and childhood but become classic RTT in adolescence after a slower, protracted course.

Evidence for both neuronal cell autonomous and nonautonomous effects of methyl-CpG-binding protein 2 in the cerebral cortex of female mice with Mecp2 mutation

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene MECP2, encoding methyl-CpG-binding protein 2 (MeCP2). Few studies have explored dendritic morphology phenotypes in mouse models of RTT and none have determined whether these phenotypes in affected females are cell autonomous or nonautonomous. Using confocal microscopy analysis we have examined the structure of dendrites and spines in the motor cortex of wild-type (WT) and Mecp2-mutant mice expressing green fluorescent protein (GFP). In Mecp2 GFP female mice age 6–7 months we found significant decreases in the density of spines, width of dendrites, size of spine heads, while increases were found in the length of spine necks, dendritic irregularities, spineless spots, and long spines. We show for the first time that a lower density of spines and smaller spine head area are phenotypes that distinguish MeCP2+ from MeCP2− dendrites in female Mecp2 GFP mice. In Mecp2 GFP male mice at three weeks of age, we found reduced spine density, thinner apical oblique dendrites and increased dendritic irregularities and long spines. Significantly, the changes affected both MeCP2− and MeCP2+ neurons, pointing to the ability of MeCP2− to impact the structure of MeCP2+ neurons. Our findings are evidence that MeCP2 deficiency results in both cell autonomous and nonautonomous changes.

Bdnf overexpression in hippocampal neurons prevents dendritic atrophy caused by Rett-associated MECP2 mutations

The expression of the methylated DNA-binding protein MeCP2 increases during neuronal development, which suggests that this epigenetic factor is crucial for neuronal terminal differentiation. We evaluated dendritic and axonal development in embryonic day-18 hippocampal neurons in culture by measuring total length and counting branch point numbers at 4 days in vitro, well before synapse formation. Pyramidal neurons transfected with a plasmid encoding a small hairpin RNA (shRNA) to knockdown endogenous Mecp2 had shorter dendrites than control untransfected neurons, without detectable changes in axonal morphology. On the other hand, overexpression of wildtype (wt) human MECP2 increased dendritic branching, in addition to axonal branching and length. Consistent with reduced neuronal growth and complexity in Rett syndrome (RTT) brains, overexpression of human MECP2 carrying missense mutations common in RTT individuals (R106W or T158M) reduced dendritic and axonal length. One of the targets of MeCP2 transcriptional control is the Bdnf gene. Indeed, endogenous Mecp2 knockdown increased the intracellular levels of BDNF protein compared to untransfected neurons, suggesting that MeCP2 represses Bdnf transcription. Surprisingly, overexpression of wt MECP2 also increased BDNF levels, while overexpression of RTT-associated MECP2 mutants failed to affect BDNF levels. The extracellular BDNF scavenger TrkB-Fc prevented dendritic overgrowth in wt MECP2-overexpressing neurons, while overexpression of the Bdnf gene reverted the dendritic atrophy caused by Mecp2-knockdown. However, this effect was only partial, since Bdnf increased dendritic length only to control levels in mutant MECP2-overexpressing neurons, but not as much as in Bdnf-transfected cells. Our results demonstrate that MeCP2 plays varied roles in dendritic and axonal development during neuronal terminal differentiation, and that some of these effects are mediated by autocrine actions of BDNF.

A Multiplexed ARMS-PCR Approach for the Detection of Common MECP2 Mutations

Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder, is caused mainly by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). Although more than 200 different MECP2 mutations have been identified throughout the gene, 7 of those (p.R133C, p.T158M, p.R168X, p.R255X, p.R270X, p.R294X, and p.R306C) account for up to two-thirds of pathogenic mutations in RTT patients. A rapid and efficient screening strategy for these mutations can be used as a preliminary step for genetic diagnosis of RTT. The current protocols used for this purpose are of high cost and require special equipment. We have designed a simpler multiplex amplification refractory mutation system (ARMS)-PCR strategy that allows identification of these common MECP2 mutant alleles in four PCR reactions. The assay was tested in 14 RTT patients who were previously genotyped using PCR-restriction fragment length polymorphism and DNA sequencing. A complete concordance was observed between the results of the two methods. The multiplex ARMS-PCR does not require any special equipment, and it provides rapid, reproducible, and cost-effective detection of common MECP2 mutations. The assay can be carried out efficiently in a standard molecular genetics laboratory and suitable as a preliminary screen for all patients with RTT diagnosis.

A possible association between genotypes and early signs of Rett disorder

Introduction: An apparently normal early development was one of the initial criteria for classic Rett syndrome. However, several investigators considered Rett syndrome to be a developmental disorder manifesting very soon after birth. Different types and positions of the MECP2 mutations and a variable pattern of x-inactivation contribute to the range of severity of this disorder. Methods: Videos of 32 Rett cases of various genotypes were carefully assessed for movements, posture and behaviour during the first 6 months of life. All signs, which deviated from the normal standard, were recorded meticulously. Special attention was paid to the face, the hands and to body movements. Results: A detailed analysis clearly demonstrated the following early abnormal signs: long lasting tongue protrusion, asymmetric eye opening and closing, bursts of abnormal facial expressions, bizarre smile, abnormal finger movements, hand stereotypies, an abnormal quality of general movements, postural stiffness, tremor and stereotyped body movements. R168X and R255 mutations were associated with the earliest onset (first days of life) of these abnormal signs. Discussion: Rett disorder is manifest within the first months of life. Those MECP2 mutations associated with the most severe clinical phenotype were also associated with an earlier onset of abnormal signs. (FWF P19581-B02, Lanyar Foundation P325).

The first missense mutation causing Rett syndrome specifically affecting the MeCP2_e1 isoform

We report the identification of the first de novo mutation at a highly conserved residue within the polyalanine stretch in the N-terminal region of the brain-dominant protein isoform MeCP2_e1 in a girl with classical Rett syndrome. The missense mutation, p.Ala2Val, leads to severe developmental delay, microcephaly, no language, severe epilepsy, and cognitive impairment. To evaluate the pathogenic potentials of the MECP2 mutation specific to the MeCP2_e1 isoform detected in this patient, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to generate a fusion protein with c-myc, and constructs were transfected in COS7 cells. In vitro studies demonstrated that, like wild-type MeCP2e_1, the N-terminal mutant is localized in the nucleus. Neither transcriptional nor translational effect on the MeCP2_e2 isoform was observed in fibroblasts from the p.Ala2Val patient, suggesting that MeCP2_e1 is involved in other functional process. These data suggest the important involvement of the N-terminus in the function of MeCP2 protein, and provide further evidence for the major impact of a specific MeCP2e_1 deficiency in the development of intellectual processing.

A thorough MECP2 mutation analysis

To the Editor: With great interest we have read the short report of Takahashi et al. entitled, ‘Skewed X chromosome inactivation failed to explain the normal phenotype of a carrier female with MECP2 mutation resulting in Rett syndrome’. The report describes a novel missense MECP2 mutation (P.A447T) detected in a girl with Rett syndrome and her asymptomatic carrier mother. Results from X-chromosome inactivation (XCI) studies performed on DNA from the mother showed a non-random XCI pattern. Further investigation showed that the mother’s predominantly active X-chromosome harbours the mutant allele. The authors speculate how these results could be correlated to the carrier mother’s normal phenotype and her daughter’s Rett phenotype. We consider that several relevant investigations are missing – especially to justify the title of this report. The authors need to address further genetic aspects before proposing a functional pathogenic nature of A447T. We lack information regarding the MECP2 mutation analysis. Was the Rett patient tested according to current knowledge about MECP2 mutations including testing for mutations in exon 1 or large deletions/duplications in the MECP2 gene? (1–4) If present, it could explain the Rett syndrome phenotype and then A447T would most likely be a rare variation. Are there any relatives in this family (siblings to the patient, to her mother or grandparents) who could be tested? Finding A447T in other family members (especially males) with normal phenotype would be a strong indication of a rare variation. What do protein substitution programs predict? Is A447T predicted to be a tolerated amino acids shift in MeCP2? It has to be taken into consideration that no other pathogenic missense mutations in the C-terminal of MeCP2 have been published in a Rett patient.

Brain Lipid Analysis in Mice with Rett Syndrome

Rett syndrome (RS) is an X-linked neurodevelopmental disorder mostly involving mutations in the gene for methyl-CpG-binding protein 2 (MECP2). Ganglioside abnormalities were previously found in cerebrum and cerebellum in RS patients. We evaluated total lipid distribution in cerebrum/brainstem, hippocampus, and cerebellum in male mice carrying either the Mecp2 (tm1.1Bird) knockout mutation or the Mecp2 (308/y) deletion mutation. The concentration of the neuronal enriched ganglioside GD1a was significantly lower in the cerebrum/brainstem of Mecp2 (tm1.1Bird) mice than in that of age matched controls, but was not reduced in the Mecp2 (308/y) mice. No other differences in brain lipid content, including myelin-enriched cerebrosides, were detected in mice with either type of Mecp2 mutation. These findings indicate that the poor motor performance previously reported in the RS mutant mice is not associated with major brain lipid abnormalities and that most previous brain lipid abnormalities observed in RS patients were not observed in the Mecp2 (tm1.1Bird) or the Mecp2 (308/y) RS mice.

Reciprocal co-regulation of EGR2 and MECP2 is disrupted in Rett syndrome and autism

Mutations in MECP2, encoding methyl-CpG-binding protein 2 (MeCP2), cause the neurodevelopmental disorder Rett syndrome (RTT). Although MECP2 mutations are rare in idiopathic autism, reduced MeCP2 levels are common in autism cortex. MeCP2 is critical for postnatal neuronal maturation and a modulator of activity-dependent genes such as Bdnf (brain-derived neurotropic factor) and JUNB. The activity-dependent early growth response gene 2 (EGR2), required for both early hindbrain development and mature neuronal function, has predicted binding sites in the promoters of several neurologically relevant genes including MECP2. Conversely, MeCP2 family members MBD1, MBD2 and MBD4 bind a methylated CpG island in an enhancer region located in EGR2 intron 1. This study was designed to test the hypothesis that MECP2 and EGR2 regulate each other’s expression during neuronal maturation in postnatal brain development. Chromatin immunoprecipitation analysis showed EGR2 binding to the MECP2 promoter and MeCP2 binding to the enhancer region in EGR2 intron 1. Reduction in EGR2 and MeCP2 levels in cultured human neuroblastoma cells by RNA interference reciprocally reduced expression of both EGR2 and MECP2 and their protein products. Consistent with a role of MeCP2 in enhancing EGR2, Mecp2-deficient mouse cortex samples showed significantly reduced EGR2 by quantitative immunofluorescence. Furthermore, MeCP2 and EGR2 show coordinately increased levels during postnatal development of both mouse and human cortex. In contrast to age-matched Controls, RTT and autism postmortem cortex samples showed significant reduction in EGR2. Together, these data support a role of dysregulation of an activity-dependent EGR2/MeCP2 pathway in RTT and autism.

A novel hypomorphic MECP2 point mutation is associated with a neuropsychiatric phenotype

The MECP2 gene on Xq28 encodes a transcriptional repressor, which binds to and modulates expression of active genes. Mutations in MECP2 cause classic or preserved speech variant Rett syndrome and intellectual disability in females and early demise or marked neurodevelopmental handicap in males. The consequences of a hypomorphic Mecp2 allele were recently investigated in a mouse model, which developed obesity, motor, social, learning, and behavioral deficits, predicting a human neurobehavioral syndrome. Here, we describe mutation analysis of a nondysmorphic female proband and her father who presented with primarily neuropsychiatric manifestations and obesity with relative sparing of intelligence, language, growth, and gross motor skills. We identified and characterized a novel missense mutation (c.454C>G; p.P152A) in the critical methyl-binding domain of MeCP2 that disrupts MeCP2 functional activity. We show that a gradient of impairment is present when the p.P152A mutation is compared with an allelic p.P152R mutation, which causes classic Rett syndrome and another Rett syndrome-causing mutation, such that protein-heterochromatin binding observed by immunofluorescence and immunoblotting is wild-type > P152A > P152R > T158 M, consistent with the severity of the observed phenotype. Our findings provide evidence for very mild phenotypes in humans associated with partial reduction of MeCP2 function arising from subtle variation in MECP2.

Rett Syndrome and long‐term disorder profile

Since the identification of mutations in MECP2 in females with clinical Rett syndrome, numerous efforts have been made to understand phenotype‐genotype relationships. Most of these studies were conducted by examining the type and localization of these mutations in the gene in relation to clinical severity. It seemed unsatisfactory, in view of the age range and variable severity, to look only at the type and localization of the mutation in MECP2 in trying to establish a phenotype/genotype relationship. Describing each RTT individual after a long term follow up and grouping females with the same disorder history and same MECP2 mutation is therefore appropriate.Complete clinical and molecular data were obtained on 103 females. The guidelines for reporting manifestations common in Rett syndrome were used in the evaluation of clinical severity. The individuals were grouped according to similar disorder profiles on long‐term follow up. In a cohort of 103 females clinically diagnosed with Rett syndrome, 91 had a detectable MECP2 mutation. 60% still sit and walk. Hand use is preserved or reduced in 44%. Speech has been lost in the majority (87%). Epilepsy was problematic in about 30%. Scoliosis, severe kyphosis or a combination of both was present in 27%, needing surgery in 13%.Description of the profile of the disorder and long‐term clinical history facilitated the grouping of females with the same MECP2 mutation and a similar history. This approach will contribute more to the understanding of the ongoing pathology in Rett syndrome relative to the specific character of the involved MECP2 mutation. Some examples of these disorder profiles will be discussed.

Methyl-CpG-binding protein 2 polymorphisms and vulnerability to autism.

The methyl-binding protein gene, MECP2, is a candidate for involvement in autism through its implication as a major causative factor in Rett syndrome that has similarities to autism. Rare mutations in MECP2 have also been identified in autistic individuals. We have examined the possible broader involvement of MECP2 as a predisposing factor in the disorder. Analysis of polymorphic markers spanning the gene and comprising both microsatellites and single nucleotide polymorphisms (SNPs) by the transmission disequilibrium test in two collections of families (219 in total), one in the USA and one in the UK, has provided evidence for significant association (P = 0.009) for a three-marker SNP haplotype of MECP2 with autism/autism spectrum disorders. This association is supported by association of both Single Sequence Repeat (SSR) and SNP single markers located at the 3' end of the MECP2 locus and flanking sequence, the most significant being that of an indel marker located in intron 2 (P = 0.001 - Bonferroni corrected P = 0.006). This suggests that one or more functional variants of MECP2 existing at significant frequencies in the population may confer increased risk of autism/autism spectrum disorders and warrants further investigation in additional independent samples.

Expression of methyl CpG binding protein 2 (Mecp2) during the postnatal development of the mouse brainstem

Methyl CpG binding protein 2 (MeCP2) is a member of the methylated DNA binding protein family able to modulate the transcription of target genes. Mutations in MECP2 lead to a wide range of neurological phenotypes and the better known of these diseases is Rett Syndrome. All patients having a mutation in MECP2 are mentally retarded and most of them exhibit dysfunctions in autonomic processes that are controlled by the brainstem. Previous studies have shown that Mecp2 is developmentally and spatially regulated throughout the rodent brain but none of them investigated the brainstem. In the present study, we have quantified the levels of expression of the Mecp2 mRNA by real time PCR and MeCP2 protein by immunoquantifications, in different areas of the mouse brainstem during the postnatal development (P0, P7, P21, P35 and P55). We focused on regions of the pons and the medulla oblongata directly involved in the regulation of autonomic functions. Our results show that the expression of MeCP2 is heterogeneously expressed throughout the postnatal mouse brainstem. MeCP2 expression in each area studied is restricted to neurones. The developmental pattern is mainly characterized by a postnatal decrease of the Mecp2 mRNA and an increase of the MeCP2 protein staining level in spite of the local variability. However, we were not able to correlate the developmental expression of MeCP2 in a given area of the brainstem with autonomic dysfunctions occurring in the presence of a mutation in Mecp2.

A MECP2 mutation in a highly conserved aminoacid causing mental retardation in a male

MeCP2 is a protein that functions as a key factor in epigenetic transcriptional regulation. Mutations in MECP2 gene have been reported as being the major cause of Rett syndrome. These mutations may also cause a wide spectrum of neurological disorders in males. Here, we report the identification of the mutation p.P405L in a 19-year-old Brazilian male with mental retardation. This variant is localized in a highly conserved aminoacid from the carboxy terminal domain and may affect the protein function. Segregation analysis on the patient’s mother revealed that this is a de novo mutation and it was not identified in the control sample. The programs SIFT, PolyPhen and A-GVGD considered that the p.P405L may be damaging. Despite the high frequency of non pathogenic variants that have been identified in this gene, our data lead us to consider the p.P405L a disease-causing mutation.

Severe congenital encephalopathy caused by MECP2 null mutations in males: central hypoxia and reduced neuronal dendritic structure

Non-mosaic males with a 46,XY karyotype and a MECP2 null mutation display a phenotype of severe neonatal-onset encephalopathy that is distinctly different from Rett syndrome (RTT). To increase awareness of this rare disorder, we are reporting novel findings in a sporadic case, compare them to 16 previously reported cases and establish salient criteria for clinical diagnosis. The proband suffered from general hypotonia and hypoxia caused by hypoventilation and irregular breathing. He developed abnormal movements, seizures and electroencephalogram abnormalities. He failed to thrive and to reach any motor milestones and died at 15 months from central respiratory failure without a diagnosis. In a muscle biopsy, type II fibers were reduced in diameter, indicating central hypoxia. At autopsy, the brain was small with disproportionate reduction of the frontal and temporal lobes. Synaptophysin staining of synaptic vesicles was greatly reduced in cerebellar and spinal cord sections. Analysis of Golgi-stained pyramidal neurons from cortical layers III and V of the frontal and temporal lobes revealed drastically diminished dendritic trees. Post-mortem MECP2 mutation analysis on DNA and RNA from fibroblasts revealed a novel de novo 9-nucleotide deletion including the intron 3/exon 4 splice junction. The two nucleotides flanking the deletion form a new splice site, and the aberrantly spliced transcript lacks seven nucleotides (r.378_384delTCCCCAG), causing a frameshift and premature termination codon (p.I126fsX11). Males with congenital encephalopathy, not females with RTT, represent the true human counterpart for the commonly studied Mecp2-/y mouse model and provide unique insight into the mechanisms of MeCP2 deficiency.

Index of Suspicion

Index of Suspicion