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Genetics and mechanisms of disease in Rett syndrome
Vol. 2, No. 4 2005
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Nervous system MECHANISMS
Genetics and mechanisms of disease in Rett syndrome Francesca Mari1, Charlotte Kilstrup-Nielsen2, Franca Cambi3, Caterina Speciale1, Maria Antonietta Mencarelli1, Alessandra Renieri1,* 1
Medical Genetics, Molecular Biology Department, University of Siena, Policlinico Le Scotte, Viale Bracci, 2, 53100 Siena, Italy Dipartimento di Biologia Strutturale e Funzionale, Universita` dell’Insubria, Via A. da Giussano 12, 21052 Busto Arsizio (VA), Italy 3 Department of Neurology, University of Kentucky, Lexington, 40536 KY, USA 2
Rett syndrome is a severe X-linked neurodevelopmental disorder and the second most common cause of mental retardation in females. In addition to the classic form characterized by typical clinical manifestations, five variants have been described. Mutations in the MECP2 gene account for the majority of Rett cases, whereas genetic heterogeneity is associated with the variants. This review will focus on recently published work that reveals novel aspects of the molecular biology of Rett syndrome.
Introduction Rett syndrome (RTT, OMIM#312750) is a sporadic X-linked neurodevelopmental disorder in girls. In the classic form, psychomotor regression begins a few months after an apparently normal development [1]. The phenotype consists of MICROCEPHALY (see Glossary), STEREOTYPIC HAND MOVEMENTS (see Glossary), loss of speech, AUTISM (see Glossary), SEIZURES (see Glossary) and SOMATIC HYPOEVOLUTISM (see Glossary). Five variants have been described: (i) preserved speech variant (PSV) with some speech recovery; (ii) early onset seizures variant with seizures at onset; (iii) ‘forme fruste’ with a milder and incomplete clinical course; (iv) congenital variant lacking the normal perinatal period; and (v) late regression variant, which is rare and still controversial [2].
*Corresponding author: A. Renieri ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2005.11.001
Section Editors: Andrey Mazarati – Department of Pediatrics, UCLA, USA Claude Wasterlain – Department of Cardiology, UCLA, USA To date, two genes are linked to this disorder. De novo mutations in the MECP2 gene (GenBank accession nos. NM_004992 and BX_538060) are found in 80–90% of classic RTT patients and in 20–40% of patients with RTT variants [3,4]. Some MECP2 mutations cause neonatal ENCEPHALOPATHY and MENTAL RETARDATION in males [5]. Mutations in the CDKL5 (GenBank accession no. NM_003159) gene, located in Xp22, are associated with the early onset seizures variant [6,7].
Main body text The MECP2 gene and its function The methyl-CpG-binding protein 2 (MeCP2) is a transcriptional repressor that binds methylated DNA through the highly conserved methyl-CpG-binding domain (MBD) whereas the transcription repression domain (TRD) mediates transcriptional repression by interacting with corepressor complexes [8–10]. MeCP2-mediated gene silencing occurs through chromatin modifications mediated by MeCP2 interactions with complexes containing Sin3A, histone deacetylases (HDAC), histone methyl transferases (HMT) and the SWI/SNF chromatin remodeling activities (Fig. 1a) [9–12]. Furthermore, the interaction of MeCP2 with the basal transcriptional machinery suggests its involvement in a chromatin-independent transcriptional repression (Fig. 1b) [13]. More recently, MeCP2 has been implicated in maintaining www.drugdiscoverytoday.com
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Glossary Autism: disorder beginning in childhood, marked by the presence of markedly abnormal or impaired development in social interaction and communication and a markedly restricted repertoire of activity and interest. Manifestations of the disorder vary greatly depending on the developmental level and chronological age of the individual (DSM-IV). Encephalopathy: generalized brain dysfunction marked by varying degrees of impairment of speech, cognition, orientation and arousal. In mild instances, brain dysfunction might be evident only during specialized neuropsychiatric testing; in severe instances the patient might be unresponsive even to unpleasant stimuli. Mental retardation: significant subaverage general intellectual functioning that is accompanied by limitations in adaptive functioning in at least two of the following skill areas: communication, self-care, home living, social/interpersonal skills, use of community resources, selfdirection, functional academic skills, work, leisure, health and safety. The onset must occur before age 18. General intellectual functioning is defined by the intelligence quotient, IQ. In general an IQ of 70 or below indicates mental retardation (mild = 50/55–70; moderate = 35/40–50/ 55; severe = 20/25–35/40; profound = below 20/25); an IQ of 70–85 signifies borderline intellectual functioning. Adaptive functioning refers to how effectively individuals cope with common life demands and these are less objective measured. Microcephaly: circumference of the head smaller than the average for age and gender based on standardized charts. Head size is assessed by measuring maximum circumference of the head or occipital–frontal circumference (OFC), and the term microcephaly is used when the head is less than that of 97–99% of the population, or below the third centile. Microcephaly might be congenital (present at birth) or it might develop in the first few years of life. Seizure: clinical or subclinical disturbances of cortical function due to a sudden, abnormal, excessive and disorganized discharge of brain cells. Clinical manifestations include abnormal motor, sensory and psychic phenomena. Recurrent seizures are usually referred to as epilepsy or ‘seizure disorder.’ Somatic hypoevolutism: retardation of somatic development from the average values for a specific age and sex based on standardized charts. The term is used when weight and length are less than that of 97– 99% of the population, or below the third centile. Stereotypic movements: inappropriate, persistent repetition of particular bodily postures, actions, or speech patterns. These are typically involuntary, rhythmic, coordinated and purposeless movements, postures, or vocalizations that might appear ritualistic or purposeful in nature. Stereotypes might be associated with a variety of neurologic and behavioral disorders.
imprinting through chromatin looping (Fig. 1c) [14]. MECP2 is almost ubiquitously expressed; therefore, it has been considered to be a global transcriptional repressor [15]. However, recently MeCP2 was shown to have a novel role in the dynamic regulation of gene silencing (see subsequent section). MeCP2 is encoded by a four-exon gene located in Xq28 [16]. Although the gene is almost ubiquitously expressed, its expression is high in brain and occurs with a temporal and spatial pattern that correlates with neuronal maturation [17]. Two MeCP2 isoforms that differ in the N-terminus are generated by alternative splicing of exon 2 [18,19]. The first identified isoform, MeCP2-e2 (MeCP2b or MeCP2A) uses a translational start site within exon 2, whereas the new isoform, MeCP2-e1 (MeCP2a or MeCP2B) derives from an 420
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Figure 1. Models of transcription repression by MeCP2. Repression of transcription might be a result of several mechanisms. (a) Chromatin-dependent transcriptional repression is mediated by the interaction of MeCP2 with histone deacetylases complex (HDAC), Switch Independent 3A (SIN3A), histone methyl transferases (HMT) and SWI/SNF (mating type switching/sucrose nonfermenting) complex. (b) Chromatin-independent transcriptional repression through the MeCP2-mediated inhibition of the basal transcriptional machinery probably through interaction and displacement of TFIIB (general transcription factor IIB). (c) MeCP2-mediated chromatin remodeling involves the formation of a chromatin higher order loop at repressed loci.
mRNA in which exon 2 is excluded and a new in-frame ATG located within exon 1 is used. Interestingly, MeCP2e1 is the predominant form in the brain [19]. It remains to be determined whether the two isoforms are functionally similar or have separate roles in gene expression. MeCP2-e2 can rescue the phenotype of Mecp2-null mice, thus, it would appear that the two proteins have redundant functions [20].
Genetics RTT is an X-linked dominant disorder mainly caused by de novo mutations in the MECP2 gene (reviewed in Ref. [4]). To date, only a few familial cases have been reported. In these cases, the MECP2 mutation was either present in the asymptomatic mother or absent in both parents owing to germline mosaicism [21]. The lack of phenotypic expression in the unaffected carrier mothers was shown to correlate with
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skewed X-inactivation [22]. Skewed X-inactivation has also been investigated as a mechanism responsible for phenotypic heterogeneity in RTT patients (reviewed in Ref. [4]). Of classic RTT patients, 80–90% carry de novo MECP2 mutations, which consist of either point mutations or small deletions (http://www.biobank.unisi.it, http://www.MeCP2. org.uk/) [3]. On the basis of the effect on the protein, mutations can be classified as: (i) early truncating mutations in the MBD and TRD domains leading to complete loss of MeCP2 function; (ii) late truncating mutations in the C-terminal domain leaving the MBD and TRD domains intact; and (iii) missense mutations usually clustered in the MBD and TRD domains. The identification of a new MECP2 isoform has had practical implications for molecular diagnosis. Exon 1, which was previously excluded from mutation screening, has been rapidly included in the MECP2 routine molecular analysis of RTT patients. On the basis of the current data, it appears however that mutations in MECP2 exon 1 are not a common cause of RTT [23]. In classic RTT patients every type of mutation has been found; by contrast, PSV patients carry only late truncating or missense mutations [3,22]. Therefore, the presence of late truncating or missense mutation, along with specific clinical findings might help to predict a more favorable course of the disease [24].
Disease mechanisms Most MECP2 mutations were predicted to cause either total or partial loss of function with few exceptions [4,25,26]. Recently, however, MECP2 overexpression was found in males with mental retardation (MR) [27]. One of the unresolved questions in RTT onset is how the loss of function of a ubiquitously expressed gene, like MECP2, predominantly, although not exclusively, causes a neurological phenotype. Constitutive and conditional Mecp2-null mice both develop RTT-like phenotypes indicating that the behavioral and neuropathological abnormalities observed in these mice are due to loss of MeCP2 expression and function in the nervous system [28,29]. These mice were also exploited to identify genes whose expression was differentially regulated in response to loss of Mecp2 [30–32]. Surprisingly, the expression of only a few genes was found to be differentially regulated in Mecp2-deficient brains [32]. Similar results were obtained in post-mortem brain tissues of RTT patients [30]. These findings do not support the hypothesis that MeCP2 is a general transcriptional repressor affecting a wide range of genes. There are several possible explanations for these unexpected results. First, there might be functional redundancy and other members of the MBD protein family might compensate for loss of MeCP2 function. Second, the microarray technology might not be sufficiently sensitive to detect changes in the expression of only a small subset of cells
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and/or very subtle changes in the expression of only a few genes. In the past couple of years, MeCP2 targets have been identified using the candidate gene approach [14,33–36]. The first gene shown to be repressed by MeCP2 was the gene encoding for brain-derived neurotrophic factor (BDNF), a protein that has essential functions for neuronal plasticity, learning and memory. In basal conditions, BDNF expression is repressed by MeCP2 bound to its promoter; upon membrane depolarization, phosphorylated MeCP2 dissociates from the promoter and BDNF expression is induced by binding of CREB [33,34]. In MeCP2-deficient neurons, the basal levels of BDNF are twofold higher than those of wild-type neurons, whereas there are no significant changes in activityinduced levels of BDNF expression (Fig. 2). Recently, Horike et al. [14] reported that loss of imprinting of a maternally expressed gene, DLX5 (GenBank accession no. NM_005221), occurs in both Mecp2-null mice and in lymphoblastoid cell lines obtained from RTT patients [37]. MeCP2 was shown to be essential for the formation of a silent chromatin structure at the Dlx5 locus by histone methylation and through the formation of a chromatin loop. Deregulation of a single gene, Dlx5, can provide a unique mechanism for some clinical manifestations of the disease. Dlx5 regulates GABA neurotransmission and osteogenesis; therefore, alterations in Dlx5 expression can account for epilepsy, osteoporosis and somatic hypoevolutism observed in RTT girls (Fig. 2). Other genes that were subsequently found to be regulated by MeCP2 in brain tissues are the ubiquitin protein ligase 3A (UBE3A – GenBank accession no. NM_130838) and the glucocorticoid inducible genes, Sgk1 (GenBank accession no. NM_005627) and Fkbp5 (GenBank accession no. NM_004117) [35,36]. In the absence of MeCP2, an approximate twofold change can be observed in the expression of UBE3A, Sgk1 and Fkbp5 (Fig. 2). In summary, these findings demonstrate a role of MeCP2 in the regulation of a small subset of genes. These data support the current hypothesis that RTT onset is due to deregulation of genes that have crucial importance for the nervous system.
Can MECP2 dosage explain the phenotypic diversity between RTT and MR? Although germline MECP2 mutations were considered to be lethal in males, we first reported a family in which an MECP2 mutation was segregated in males with recessive X-linked mental retardation (XLMR) and spasticity [5]. Subsequently, several investigators have reported MECP2 mutations in males with nonfatal, nonprogressive encephalopathy, males with nonspecific X-linked mental retardation (MRX) and males with language disorder and schizophrenia (reviewed in Ref. [4]). To date, only late truncating and missense mutations in the MBD and TRD were identified in these X-linked recessive mental retardation cases, whereas early truncating www.drugdiscoverytoday.com
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Figure 2. Disease mechanisms. Loss of MeCP2 leads to overexpression of specific genes: BDNF, DLX5, Sgk1, Fkbp5 and antisense UBE3A. Overexpression of BDNF might be responsible for impairment of brain development, learning and memory. The overexpression of DLX5 might induce glutamic acid decarboxylase expression which might result in abnormal differentiation of GABAergic neurons. Overexpression of DLX5 might also be responsible for bone impairment present in RTT patient, such as osteoporosis and somatic hypoevolutism. Sgk1 overexpression might lead to cell survival and alterations in neuronal excitability by activating specific ion channels. Overexpression of Fkbp5, a glucocorticoid receptor that regulates the cochaperone of Hsp90, might lead to impairment of steroid hormone signaling. Finally, the overexpression of antisense UBE3A results in underexpression of UBE3A, the gene responsible for the Angelman syndrome, possibly explaining the Angelman-like features present in patients with RTT syndrome [35].
mutations, which are associated with severe encephalopathy in male siblings of RTT females, were not found. Recent advances might explain the previously reported phenotypic diversity. Recently, duplications of the Xq28 region encompassing the MECP2 gene were shown to segregate with mental retardation and progressive neurological symptoms in males [27]. The authors demonstrated that the gene dosage mechanism correlates with disease presentation and that skewed X-inactivation accounts for the absence of disease in carrier females. On the basis of these findings, it is possible to propose a model in which different balancing of MECP2 expression might be the cause of phenotypic diversity (Fig. 3). A gene-dosage-sensitive mechanism was confirmed in Mecp2 transgenic mice, which develop a neurological phenotype of varying severity depending on the Mecp2 gene copy number [38]. Similar results were observed in Mecp2null mice after Mecp2 gene replacement into the tau locus [20]. Taken together, these data provide firm evidence that the central nervous system is extremely sensitive to MECP2 expression levels and that tight regulation of MECP2 expression is crucial in pre- and postnatal brain development and function. Laccone et al. [39] showed that an intraperitoneal injection of the TAT–MeCP2 recombinant protein into knockout mice results in biologically active Mecp2 in the 422
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brain. Although these preliminary results are encouraging and could lead to therapeutic options for this debilitating disorder, the importance of tight regulation of MECP2 expression levels needs to be considered carefully in designing treatment strategies based on gene correction of RTT.
Is RTT a genetically heterogeneous disorder? Recently, point mutations in the cyclin-dependent kinase like 5 (CDKL5) gene (previously known as STK9), located in Xp22, have been identified in female patients with the ‘early onset seizures variant’ of RTT [6,21,40–42]. Mutations
Figure 3. MeCP2 dosage balancing. According to a balancing model, mutations that lead to increased MeCP2 expression cause mental retardation in males and are silent in females because of skewed X inactivation. Mutations that cause loss of MeCP2 cause RTT in females and severe perinatal encephalopathy in males. M: males; F: females.
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Figure 4. CDKL5–MeCP2 interaction. The C-terminal portion of MeCP2 directly interacts with the middle part of CDKL5. CDKL5 is a protein kinase that can phosphorylate itself and MeCP2. Phosphorylation participates in the regulation of MeCP2 function; therefore, defects in CDKL5 could affect MeCP2 function through changes in phosphorylation.
in this gene have not been identified in the classic form and in other RTT variants ([6,43], A. Renieri et al., unpublished). The observation that mutations in MECP2 and CDKL5 cause similar phenotypes suggested that these genes might participate in the same molecular pathways. Mari et al. [7] showed that MeCP2 and CDKL5 have an overlapping temporal and spatial expression profile during neuronal maturation and synaptogenesis and that they physically interact. The interaction was shown to require a portion of the Cterminal domain of MeCP2, suggesting that mutations in this region might be involved in RTT onset owing to loss of interaction between the two proteins. Furthermore, it was shown that the kinase activity of CDKL5 can cause both autophosphorylation and MeCP2 phosphorylation (Fig. 4). These findings further support the hypothesis that the function of the two genes is tightly linked and give a preliminary explanation to the involvement of CDKL5 in RTT onset. Phosphorylation of MeCP2 has a crucial role in the regulation of BDNF (GenBank accession no. NM_170735) gene expression; therefore, it can be speculated that defects in MeCP2 phosphorylation caused by loss of CDKL5 function result in changes in BDNF gene expression. Additional studies are necessary to determine the complex mechanisms by which CDKL5 causes only one RTT variant and why this phenotype is always characterized by an early appearance of seizures [6,7,40–42]. In addition to point mutations, chromosomal rearrangements at the CDKL5 locus have been associated with other neurological phenotypes. In two unrelated female patients with West syndrome (infantile spasm syndrome X-linkedISSX), a de novo balanced X-autosome translocation was
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shown to disrupt the CDKL5 gene [44]. In a male patient with X-linked retinoschisis (XLRS) and seizures, which are not typically associated with XLRS, a large deletion involving at least the last codon of the CDKL5 gene and the flanking RS1 gene (GenBank accession no. NM_000330) was identified [45]. From these descriptions, it appears that the phenotype associated with CDKL5 gross rearrangements is more variable than that associated with point mutations and might be at least in part explained by alterations in contiguous genes. However, in all cases of CDKL5 mutations, a seizure disorder appears to be a prominent and integral manifestation of the syndrome. Until recently, RTT was considered a monogenic disorder caused by mutations in the MECP2 gene. Although the data reviewed in this article demonstrate a greater genetic complexity and heterogeneity in RTT, MECP2 is likely to be the gene primarily responsible for classic RTT cases (Table 1). The MECP2-negative classic RTT cases could be caused by mutations in the noncoding regulatory regions and intronic sequences of MECP2. Recently, mutations affecting MECP2 splicing were reported and they point to the importance of screening MeCP2 protein and RNA in MECP2-negative RTT cases [46]. By contrast, a high percentage of patients with variants of RTT do not have MECP2 mutations, leaving open the question of what is the molecular defect in these patients. Defining the molecular defects in the RTT variants is going to be challenging because there are fewer patients with RTT variants than with the classic form. In addition, the clinical diagnosis of a specific RTT variant can be difficult and is better accomplished in specialized centers. According to the published data, 50% of PSV variant cases are MECP2-negative and 50% of the early onset seizures variant cases are CDKL5 negative (Table 1). Some of these cases could still carry mutations in noncoding regions of either MECP2 or CDKL5. The ‘forme fruste’ characterized by a milder and incomplete clinical course might represent a genetically heterogeneous group, possibly linked to novel gene(s). Interestingly, several rearrangements have been reported in RTT-like phenotypes, raising the possibility of novel candidate genes contained in these regions ([47] and reviewed in Ref. [4]).
Table 1. Mutation detection rate in known genes MECP2 (%) CDKL5 (%) Unknown (%) Classic form
90
0
10
Preserved speech variant 50
0
50
Forme fruste
10
0
90
Congenital variant
10
0
90
Late regression variant
?
?
?
Early onset variant
0
50
50
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In conclusion, we believe that RTT, especially in its variants, is a heterogeneous disorder and would expect that additional genes might soon be discovered as being the cause of RTT.
Research perspectives Since the discovery of MECP2 as the causative gene of RTT, the spectrum of clinical manifestations associated with MECP2 mutations has expanded enormously. In addition, a novel gene, namely CDKL5, has been identified as the cause of some RTT variants and target genes that are dysregulated by loss of MeCP2 have been characterized. In addition to the original view of MeCP2 as a global repressor of gene expression, a more dynamic function of this protein in transcriptional regulation has emerged. It has also been shown that MeCP2 can cause disease through a gene-dosage-sensitive mechanism and that overexpression has deleterious consequences, especially in males. Future work should be aimed at ‘filling the gap’ for the MECP2-negative RTT cases and searching for intronic mutations causing miss-splicing of MECP2, mutations in regulatory regions affecting levels of expression, screening target genes already identified and genes that will be identified in the future. It is conceivable that mutations in target genes also account for some RTT variants. Significant effort should be devoted to the identification of other genes that are dysregulated by MECP2 mutations and duplications. Are the same targets affected or is gene dosage acting through a separate mechanism? It will be necessary to determine the downstream effects caused by altered expression of BDNF and UBE3A and other genes that will be identified and to investigate the specific phenotypic manifestations that are caused by dysregulation of these genes. These studies will be crucially important for the development of therapies aimed at correcting the phenotype. Although gene replacement is an option, this might be problematic in light of the deleterious effects of gene dosage and of the timing of gene therapy. Potentially useful treatments might be agents that correct the expression of target genes, at least in temporally defined periods of development.
Acknowledgements This work was supported by Telethon grants GGP02372A and GTF02006, by the Emma and Ernesto Rulfo Foundation, by the Ministero della Salute (Progetti di Ricerca Finalizzata, D.L. 502/92-2003), by MIUR (FIRB 01) and by the University of Siena (PAR 2001 and PAR 2002) to A.R. and by RSRF Research Grant to C.K-N.
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Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Nervous system MECHANISMS
Genetics and mechanisms of disease in Rett syndrome Francesca Mari1, Charlotte Kilstrup-Nielsen2, Franca Cambi3, Caterina Speciale1, Maria Antonietta Mencarelli1, Alessandra Renieri1,* 1
Medical Genetics, Molecular Biology Department, University of Siena, Policlinico Le Scotte, Viale Bracci, 2, 53100 Siena, Italy Dipartimento di Biologia Strutturale e Funzionale, Universita` dell’Insubria, Via A. da Giussano 12, 21052 Busto Arsizio (VA), Italy 3 Department of Neurology, University of Kentucky, Lexington, 40536 KY, USA 2
Rett syndrome is a severe X-linked neurodevelopmental disorder and the second most common cause of mental retardation in females. In addition to the classic form characterized by typical clinical manifestations, five variants have been described. Mutations in the MECP2 gene account for the majority of Rett cases, whereas genetic heterogeneity is associated with the variants. This review will focus on recently published work that reveals novel aspects of the molecular biology of Rett syndrome.
Introduction Rett syndrome (RTT, OMIM#312750) is a sporadic X-linked neurodevelopmental disorder in girls. In the classic form, psychomotor regression begins a few months after an apparently normal development [1]. The phenotype consists of MICROCEPHALY (see Glossary), STEREOTYPIC HAND MOVEMENTS (see Glossary), loss of speech, AUTISM (see Glossary), SEIZURES (see Glossary) and SOMATIC HYPOEVOLUTISM (see Glossary). Five variants have been described: (i) preserved speech variant (PSV) with some speech recovery; (ii) early onset seizures variant with seizures at onset; (iii) ‘forme fruste’ with a milder and incomplete clinical course; (iv) congenital variant lacking the normal perinatal period; and (v) late regression variant, which is rare and still controversial [2].
*Corresponding author: A. Renieri ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2005.11.001
Section Editors: Andrey Mazarati – Department of Pediatrics, UCLA, USA Claude Wasterlain – Department of Cardiology, UCLA, USA To date, two genes are linked to this disorder. De novo mutations in the MECP2 gene (GenBank accession nos. NM_004992 and BX_538060) are found in 80–90% of classic RTT patients and in 20–40% of patients with RTT variants [3,4]. Some MECP2 mutations cause neonatal ENCEPHALOPATHY and MENTAL RETARDATION in males [5]. Mutations in the CDKL5 (GenBank accession no. NM_003159) gene, located in Xp22, are associated with the early onset seizures variant [6,7].
Main body text The MECP2 gene and its function The methyl-CpG-binding protein 2 (MeCP2) is a transcriptional repressor that binds methylated DNA through the highly conserved methyl-CpG-binding domain (MBD) whereas the transcription repression domain (TRD) mediates transcriptional repression by interacting with corepressor complexes [8–10]. MeCP2-mediated gene silencing occurs through chromatin modifications mediated by MeCP2 interactions with complexes containing Sin3A, histone deacetylases (HDAC), histone methyl transferases (HMT) and the SWI/SNF chromatin remodeling activities (Fig. 1a) [9–12]. Furthermore, the interaction of MeCP2 with the basal transcriptional machinery suggests its involvement in a chromatin-independent transcriptional repression (Fig. 1b) [13]. More recently, MeCP2 has been implicated in maintaining www.drugdiscoverytoday.com
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Glossary Autism: disorder beginning in childhood, marked by the presence of markedly abnormal or impaired development in social interaction and communication and a markedly restricted repertoire of activity and interest. Manifestations of the disorder vary greatly depending on the developmental level and chronological age of the individual (DSM-IV). Encephalopathy: generalized brain dysfunction marked by varying degrees of impairment of speech, cognition, orientation and arousal. In mild instances, brain dysfunction might be evident only during specialized neuropsychiatric testing; in severe instances the patient might be unresponsive even to unpleasant stimuli. Mental retardation: significant subaverage general intellectual functioning that is accompanied by limitations in adaptive functioning in at least two of the following skill areas: communication, self-care, home living, social/interpersonal skills, use of community resources, selfdirection, functional academic skills, work, leisure, health and safety. The onset must occur before age 18. General intellectual functioning is defined by the intelligence quotient, IQ. In general an IQ of 70 or below indicates mental retardation (mild = 50/55–70; moderate = 35/40–50/ 55; severe = 20/25–35/40; profound = below 20/25); an IQ of 70–85 signifies borderline intellectual functioning. Adaptive functioning refers to how effectively individuals cope with common life demands and these are less objective measured. Microcephaly: circumference of the head smaller than the average for age and gender based on standardized charts. Head size is assessed by measuring maximum circumference of the head or occipital–frontal circumference (OFC), and the term microcephaly is used when the head is less than that of 97–99% of the population, or below the third centile. Microcephaly might be congenital (present at birth) or it might develop in the first few years of life. Seizure: clinical or subclinical disturbances of cortical function due to a sudden, abnormal, excessive and disorganized discharge of brain cells. Clinical manifestations include abnormal motor, sensory and psychic phenomena. Recurrent seizures are usually referred to as epilepsy or ‘seizure disorder.’ Somatic hypoevolutism: retardation of somatic development from the average values for a specific age and sex based on standardized charts. The term is used when weight and length are less than that of 97– 99% of the population, or below the third centile. Stereotypic movements: inappropriate, persistent repetition of particular bodily postures, actions, or speech patterns. These are typically involuntary, rhythmic, coordinated and purposeless movements, postures, or vocalizations that might appear ritualistic or purposeful in nature. Stereotypes might be associated with a variety of neurologic and behavioral disorders.
imprinting through chromatin looping (Fig. 1c) [14]. MECP2 is almost ubiquitously expressed; therefore, it has been considered to be a global transcriptional repressor [15]. However, recently MeCP2 was shown to have a novel role in the dynamic regulation of gene silencing (see subsequent section). MeCP2 is encoded by a four-exon gene located in Xq28 [16]. Although the gene is almost ubiquitously expressed, its expression is high in brain and occurs with a temporal and spatial pattern that correlates with neuronal maturation [17]. Two MeCP2 isoforms that differ in the N-terminus are generated by alternative splicing of exon 2 [18,19]. The first identified isoform, MeCP2-e2 (MeCP2b or MeCP2A) uses a translational start site within exon 2, whereas the new isoform, MeCP2-e1 (MeCP2a or MeCP2B) derives from an 420
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Figure 1. Models of transcription repression by MeCP2. Repression of transcription might be a result of several mechanisms. (a) Chromatin-dependent transcriptional repression is mediated by the interaction of MeCP2 with histone deacetylases complex (HDAC), Switch Independent 3A (SIN3A), histone methyl transferases (HMT) and SWI/SNF (mating type switching/sucrose nonfermenting) complex. (b) Chromatin-independent transcriptional repression through the MeCP2-mediated inhibition of the basal transcriptional machinery probably through interaction and displacement of TFIIB (general transcription factor IIB). (c) MeCP2-mediated chromatin remodeling involves the formation of a chromatin higher order loop at repressed loci.
mRNA in which exon 2 is excluded and a new in-frame ATG located within exon 1 is used. Interestingly, MeCP2e1 is the predominant form in the brain [19]. It remains to be determined whether the two isoforms are functionally similar or have separate roles in gene expression. MeCP2-e2 can rescue the phenotype of Mecp2-null mice, thus, it would appear that the two proteins have redundant functions [20].
Genetics RTT is an X-linked dominant disorder mainly caused by de novo mutations in the MECP2 gene (reviewed in Ref. [4]). To date, only a few familial cases have been reported. In these cases, the MECP2 mutation was either present in the asymptomatic mother or absent in both parents owing to germline mosaicism [21]. The lack of phenotypic expression in the unaffected carrier mothers was shown to correlate with
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skewed X-inactivation [22]. Skewed X-inactivation has also been investigated as a mechanism responsible for phenotypic heterogeneity in RTT patients (reviewed in Ref. [4]). Of classic RTT patients, 80–90% carry de novo MECP2 mutations, which consist of either point mutations or small deletions (http://www.biobank.unisi.it, http://www.MeCP2. org.uk/) [3]. On the basis of the effect on the protein, mutations can be classified as: (i) early truncating mutations in the MBD and TRD domains leading to complete loss of MeCP2 function; (ii) late truncating mutations in the C-terminal domain leaving the MBD and TRD domains intact; and (iii) missense mutations usually clustered in the MBD and TRD domains. The identification of a new MECP2 isoform has had practical implications for molecular diagnosis. Exon 1, which was previously excluded from mutation screening, has been rapidly included in the MECP2 routine molecular analysis of RTT patients. On the basis of the current data, it appears however that mutations in MECP2 exon 1 are not a common cause of RTT [23]. In classic RTT patients every type of mutation has been found; by contrast, PSV patients carry only late truncating or missense mutations [3,22]. Therefore, the presence of late truncating or missense mutation, along with specific clinical findings might help to predict a more favorable course of the disease [24].
Disease mechanisms Most MECP2 mutations were predicted to cause either total or partial loss of function with few exceptions [4,25,26]. Recently, however, MECP2 overexpression was found in males with mental retardation (MR) [27]. One of the unresolved questions in RTT onset is how the loss of function of a ubiquitously expressed gene, like MECP2, predominantly, although not exclusively, causes a neurological phenotype. Constitutive and conditional Mecp2-null mice both develop RTT-like phenotypes indicating that the behavioral and neuropathological abnormalities observed in these mice are due to loss of MeCP2 expression and function in the nervous system [28,29]. These mice were also exploited to identify genes whose expression was differentially regulated in response to loss of Mecp2 [30–32]. Surprisingly, the expression of only a few genes was found to be differentially regulated in Mecp2-deficient brains [32]. Similar results were obtained in post-mortem brain tissues of RTT patients [30]. These findings do not support the hypothesis that MeCP2 is a general transcriptional repressor affecting a wide range of genes. There are several possible explanations for these unexpected results. First, there might be functional redundancy and other members of the MBD protein family might compensate for loss of MeCP2 function. Second, the microarray technology might not be sufficiently sensitive to detect changes in the expression of only a small subset of cells
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and/or very subtle changes in the expression of only a few genes. In the past couple of years, MeCP2 targets have been identified using the candidate gene approach [14,33–36]. The first gene shown to be repressed by MeCP2 was the gene encoding for brain-derived neurotrophic factor (BDNF), a protein that has essential functions for neuronal plasticity, learning and memory. In basal conditions, BDNF expression is repressed by MeCP2 bound to its promoter; upon membrane depolarization, phosphorylated MeCP2 dissociates from the promoter and BDNF expression is induced by binding of CREB [33,34]. In MeCP2-deficient neurons, the basal levels of BDNF are twofold higher than those of wild-type neurons, whereas there are no significant changes in activityinduced levels of BDNF expression (Fig. 2). Recently, Horike et al. [14] reported that loss of imprinting of a maternally expressed gene, DLX5 (GenBank accession no. NM_005221), occurs in both Mecp2-null mice and in lymphoblastoid cell lines obtained from RTT patients [37]. MeCP2 was shown to be essential for the formation of a silent chromatin structure at the Dlx5 locus by histone methylation and through the formation of a chromatin loop. Deregulation of a single gene, Dlx5, can provide a unique mechanism for some clinical manifestations of the disease. Dlx5 regulates GABA neurotransmission and osteogenesis; therefore, alterations in Dlx5 expression can account for epilepsy, osteoporosis and somatic hypoevolutism observed in RTT girls (Fig. 2). Other genes that were subsequently found to be regulated by MeCP2 in brain tissues are the ubiquitin protein ligase 3A (UBE3A – GenBank accession no. NM_130838) and the glucocorticoid inducible genes, Sgk1 (GenBank accession no. NM_005627) and Fkbp5 (GenBank accession no. NM_004117) [35,36]. In the absence of MeCP2, an approximate twofold change can be observed in the expression of UBE3A, Sgk1 and Fkbp5 (Fig. 2). In summary, these findings demonstrate a role of MeCP2 in the regulation of a small subset of genes. These data support the current hypothesis that RTT onset is due to deregulation of genes that have crucial importance for the nervous system.
Can MECP2 dosage explain the phenotypic diversity between RTT and MR? Although germline MECP2 mutations were considered to be lethal in males, we first reported a family in which an MECP2 mutation was segregated in males with recessive X-linked mental retardation (XLMR) and spasticity [5]. Subsequently, several investigators have reported MECP2 mutations in males with nonfatal, nonprogressive encephalopathy, males with nonspecific X-linked mental retardation (MRX) and males with language disorder and schizophrenia (reviewed in Ref. [4]). To date, only late truncating and missense mutations in the MBD and TRD were identified in these X-linked recessive mental retardation cases, whereas early truncating www.drugdiscoverytoday.com
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Figure 2. Disease mechanisms. Loss of MeCP2 leads to overexpression of specific genes: BDNF, DLX5, Sgk1, Fkbp5 and antisense UBE3A. Overexpression of BDNF might be responsible for impairment of brain development, learning and memory. The overexpression of DLX5 might induce glutamic acid decarboxylase expression which might result in abnormal differentiation of GABAergic neurons. Overexpression of DLX5 might also be responsible for bone impairment present in RTT patient, such as osteoporosis and somatic hypoevolutism. Sgk1 overexpression might lead to cell survival and alterations in neuronal excitability by activating specific ion channels. Overexpression of Fkbp5, a glucocorticoid receptor that regulates the cochaperone of Hsp90, might lead to impairment of steroid hormone signaling. Finally, the overexpression of antisense UBE3A results in underexpression of UBE3A, the gene responsible for the Angelman syndrome, possibly explaining the Angelman-like features present in patients with RTT syndrome [35].
mutations, which are associated with severe encephalopathy in male siblings of RTT females, were not found. Recent advances might explain the previously reported phenotypic diversity. Recently, duplications of the Xq28 region encompassing the MECP2 gene were shown to segregate with mental retardation and progressive neurological symptoms in males [27]. The authors demonstrated that the gene dosage mechanism correlates with disease presentation and that skewed X-inactivation accounts for the absence of disease in carrier females. On the basis of these findings, it is possible to propose a model in which different balancing of MECP2 expression might be the cause of phenotypic diversity (Fig. 3). A gene-dosage-sensitive mechanism was confirmed in Mecp2 transgenic mice, which develop a neurological phenotype of varying severity depending on the Mecp2 gene copy number [38]. Similar results were observed in Mecp2null mice after Mecp2 gene replacement into the tau locus [20]. Taken together, these data provide firm evidence that the central nervous system is extremely sensitive to MECP2 expression levels and that tight regulation of MECP2 expression is crucial in pre- and postnatal brain development and function. Laccone et al. [39] showed that an intraperitoneal injection of the TAT–MeCP2 recombinant protein into knockout mice results in biologically active Mecp2 in the 422
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brain. Although these preliminary results are encouraging and could lead to therapeutic options for this debilitating disorder, the importance of tight regulation of MECP2 expression levels needs to be considered carefully in designing treatment strategies based on gene correction of RTT.
Is RTT a genetically heterogeneous disorder? Recently, point mutations in the cyclin-dependent kinase like 5 (CDKL5) gene (previously known as STK9), located in Xp22, have been identified in female patients with the ‘early onset seizures variant’ of RTT [6,21,40–42]. Mutations
Figure 3. MeCP2 dosage balancing. According to a balancing model, mutations that lead to increased MeCP2 expression cause mental retardation in males and are silent in females because of skewed X inactivation. Mutations that cause loss of MeCP2 cause RTT in females and severe perinatal encephalopathy in males. M: males; F: females.
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Figure 4. CDKL5–MeCP2 interaction. The C-terminal portion of MeCP2 directly interacts with the middle part of CDKL5. CDKL5 is a protein kinase that can phosphorylate itself and MeCP2. Phosphorylation participates in the regulation of MeCP2 function; therefore, defects in CDKL5 could affect MeCP2 function through changes in phosphorylation.
in this gene have not been identified in the classic form and in other RTT variants ([6,43], A. Renieri et al., unpublished). The observation that mutations in MECP2 and CDKL5 cause similar phenotypes suggested that these genes might participate in the same molecular pathways. Mari et al. [7] showed that MeCP2 and CDKL5 have an overlapping temporal and spatial expression profile during neuronal maturation and synaptogenesis and that they physically interact. The interaction was shown to require a portion of the Cterminal domain of MeCP2, suggesting that mutations in this region might be involved in RTT onset owing to loss of interaction between the two proteins. Furthermore, it was shown that the kinase activity of CDKL5 can cause both autophosphorylation and MeCP2 phosphorylation (Fig. 4). These findings further support the hypothesis that the function of the two genes is tightly linked and give a preliminary explanation to the involvement of CDKL5 in RTT onset. Phosphorylation of MeCP2 has a crucial role in the regulation of BDNF (GenBank accession no. NM_170735) gene expression; therefore, it can be speculated that defects in MeCP2 phosphorylation caused by loss of CDKL5 function result in changes in BDNF gene expression. Additional studies are necessary to determine the complex mechanisms by which CDKL5 causes only one RTT variant and why this phenotype is always characterized by an early appearance of seizures [6,7,40–42]. In addition to point mutations, chromosomal rearrangements at the CDKL5 locus have been associated with other neurological phenotypes. In two unrelated female patients with West syndrome (infantile spasm syndrome X-linkedISSX), a de novo balanced X-autosome translocation was
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shown to disrupt the CDKL5 gene [44]. In a male patient with X-linked retinoschisis (XLRS) and seizures, which are not typically associated with XLRS, a large deletion involving at least the last codon of the CDKL5 gene and the flanking RS1 gene (GenBank accession no. NM_000330) was identified [45]. From these descriptions, it appears that the phenotype associated with CDKL5 gross rearrangements is more variable than that associated with point mutations and might be at least in part explained by alterations in contiguous genes. However, in all cases of CDKL5 mutations, a seizure disorder appears to be a prominent and integral manifestation of the syndrome. Until recently, RTT was considered a monogenic disorder caused by mutations in the MECP2 gene. Although the data reviewed in this article demonstrate a greater genetic complexity and heterogeneity in RTT, MECP2 is likely to be the gene primarily responsible for classic RTT cases (Table 1). The MECP2-negative classic RTT cases could be caused by mutations in the noncoding regulatory regions and intronic sequences of MECP2. Recently, mutations affecting MECP2 splicing were reported and they point to the importance of screening MeCP2 protein and RNA in MECP2-negative RTT cases [46]. By contrast, a high percentage of patients with variants of RTT do not have MECP2 mutations, leaving open the question of what is the molecular defect in these patients. Defining the molecular defects in the RTT variants is going to be challenging because there are fewer patients with RTT variants than with the classic form. In addition, the clinical diagnosis of a specific RTT variant can be difficult and is better accomplished in specialized centers. According to the published data, 50% of PSV variant cases are MECP2-negative and 50% of the early onset seizures variant cases are CDKL5 negative (Table 1). Some of these cases could still carry mutations in noncoding regions of either MECP2 or CDKL5. The ‘forme fruste’ characterized by a milder and incomplete clinical course might represent a genetically heterogeneous group, possibly linked to novel gene(s). Interestingly, several rearrangements have been reported in RTT-like phenotypes, raising the possibility of novel candidate genes contained in these regions ([47] and reviewed in Ref. [4]).
Table 1. Mutation detection rate in known genes MECP2 (%) CDKL5 (%) Unknown (%) Classic form
90
0
10
Preserved speech variant 50
0
50
Forme fruste
10
0
90
Congenital variant
10
0
90
Late regression variant
?
?
?
Early onset variant
0
50
50
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In conclusion, we believe that RTT, especially in its variants, is a heterogeneous disorder and would expect that additional genes might soon be discovered as being the cause of RTT.
Research perspectives Since the discovery of MECP2 as the causative gene of RTT, the spectrum of clinical manifestations associated with MECP2 mutations has expanded enormously. In addition, a novel gene, namely CDKL5, has been identified as the cause of some RTT variants and target genes that are dysregulated by loss of MeCP2 have been characterized. In addition to the original view of MeCP2 as a global repressor of gene expression, a more dynamic function of this protein in transcriptional regulation has emerged. It has also been shown that MeCP2 can cause disease through a gene-dosage-sensitive mechanism and that overexpression has deleterious consequences, especially in males. Future work should be aimed at ‘filling the gap’ for the MECP2-negative RTT cases and searching for intronic mutations causing miss-splicing of MECP2, mutations in regulatory regions affecting levels of expression, screening target genes already identified and genes that will be identified in the future. It is conceivable that mutations in target genes also account for some RTT variants. Significant effort should be devoted to the identification of other genes that are dysregulated by MECP2 mutations and duplications. Are the same targets affected or is gene dosage acting through a separate mechanism? It will be necessary to determine the downstream effects caused by altered expression of BDNF and UBE3A and other genes that will be identified and to investigate the specific phenotypic manifestations that are caused by dysregulation of these genes. These studies will be crucially important for the development of therapies aimed at correcting the phenotype. Although gene replacement is an option, this might be problematic in light of the deleterious effects of gene dosage and of the timing of gene therapy. Potentially useful treatments might be agents that correct the expression of target genes, at least in temporally defined periods of development.
Acknowledgements This work was supported by Telethon grants GGP02372A and GTF02006, by the Emma and Ernesto Rulfo Foundation, by the Ministero della Salute (Progetti di Ricerca Finalizzata, D.L. 502/92-2003), by MIUR (FIRB 01) and by the University of Siena (PAR 2001 and PAR 2002) to A.R. and by RSRF Research Grant to C.K-N.
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