Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G0/G1 phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5.
► CDKL5 enhances neuronal differentiation. ► CDKL5 arrests cell cycle of neuronal precursor cells. ► MYCN directly represses transcription of CDKL5. ► These results may help explain the neurological symptoms of RTT patients.
Rett's syndrome; CDKL5; MYCN; Neurogenesis; Differentiation
Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-d-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.
Glutamate; Glutamate Receptors Ionotropic (AMPA and NMDA); Neurological Diseases; Neurons; Protein Degradation; Protein Translocation; Serine Threonine Protein Kinase; CDKL5; Excitotoxicity
Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been described in girls with Rett-like features and early-onset epileptic encephalopathy including infantile spasms. To date, with more than 80 reported cases, the phenotype of CDKL5-related encephalopathy is better defined. The main features consist of early-onset seizures starting before 5 months of age, severe mental retardation with absent speech and Rett-like features such as hand stereotypies and deceleration of head growth. On the other hand, neuro-vegetative signs and developmental regression are rare in CDKL5 mutation patients. The CDKL5 gene encodes a serine threonine kinase protein which is characterized by a catalytic domain and a long C-terminal extension involved in the regulation of the catalytic activity of CDKL5 and in the sub-nuclear localization of the protein. To our knowledge, more than 70 different point mutations have been described including missense mutations within the catalytic domain, nonsense mutations causing the premature termination of the protein distributed in the entire open reading frame, splice variants, and frameshift mutations. Additionally, CDKL5 mutations have recently been described in 7 males with a more severe epileptic encephalopathy and a worse outcome compared to female patients. Finally, about 23 male and female patients have been identified with gross rearrangements encompassing all or part of the CDKL5 gene, with a phenotype reminiscent of CDKL5-related encephalopathy combined with dysmorphic features. Even if recent data clearly indicate that CDKL5 plays an important role in brain function, the protein remains largely uncharacterized. Phenotype-genotype correlation is additionally hampered by the relatively small number of patients described.
CDKL5; Epileptic encephalopathy; Rett syndrome
To determine the frequency of mutations in CDKL5 in both male and female patients with infantile spasms or early onset epilepsy of unknown cause, and to consider whether the breadth of the reported phenotype would be extended by studying a different patient group.
Two groups of patients were investigated for CDKL5 mutations. Group 1 comprised 73 patients (57 female, 16 male) referred to Cardiff for CDKL5 analysis, of whom 49 (42 female, 7 male) had epileptic seizure onset in the first six months of life. Group 2 comprised 26 patients (11 female, 15 male) with infantile spasms previously recruited to a clinical trial, the UK Infantile Spasms Study. Where a likely pathogenic mutation was identified, further clinical data were reviewed.
Seven likely pathogenic mutations were found among female patients from group 1 with epileptic seizure onset in the first six months of life, accounting for seven of the 42 in this group (17%). No mutations other than the already published mutation were found in female patients from group 2, or in any male patient from either study group. All patients with mutations had early signs of developmental delay and most had made little developmental progress. Further clinical information was available for six patients: autistic features and tactile hypersensitivity were common but only one had suggestive Rett‐like features. All had a severe epileptic seizure disorder, all but one of whom had myoclonic jerks. The EEG showed focal or generalised changes and in those with infantile spasms, hypsarrhythmia. Slow frequencies were seen frequently with a frontal or fronto‐temporal predominance and high amplitudes.
The spectrum of the epileptic seizure disorder, and associated EEG changes, in those with CDKL5 mutations is broader than previously reported. CDKL5 mutations are a significant cause of infantile spasms and early epileptic seizures in female patients, and of a later intractable seizure disorder, irrespective of whether they have suspected Rett syndrome. Analysis should be considered in these patients in the clinical setting.
; infantile spasms; epilepsy; Rett syndrome
Rett syndrome is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in 3 genes have been associated with this condition. Classic Rett syndrome is caused by mutations in the MECP2 gene, while variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of Rett syndrome and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons, but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types, including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation, affected by early onset seizure variant and X-linked epileptic encephalopathy, respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild type allele, that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore, the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro.
CDKL5; Rett syndrome; induced Pluripotent Stem cells (iPSC); disease modelling
Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene, whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons, but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types, including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation, affected by early onset seizure variant and X-linked epileptic encephalopathy, respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore, the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro.
CDKL5; Rett syndrome; induced pluripotent stem cells (iPSC); disease modelling
Rett syndrome (RS) is a severe neurodevelopmental disorder that contributes significantly to severe intellectual disability in females worldwide. It is caused by mutations in MECP2 in the majority of cases, but a proportion of atypical cases may result from mutations in CDKL5, particularly the early onset seizure variant. The relationship between MECP2 and CDKL5, and whether they cause RS through the same or different mechanisms is unknown, but is worthy of investigation. Mutations in MECP2 appear to give a growth disadvantage to both neuronal and lymphoblast cells, often resulting in skewing of X inactivation that may contribute to the large degree of phenotypic variation. MeCP2 was originally thought to be a global transcriptional repressor, but recent evidence suggests that it may have a role in regulating neuronal activity dependent expression of specific genes such as Hairy2a in Xenopus and Bdnf in mouse and rat.
In the last few years, the X-linked serine/threonine kinase cyclin-dependent kinase-like 5 (CDKL5) has been associated with early-onset epileptic encephalopathies characterized by the manifestation of intractable epilepsy within the first weeks of life, severe developmental delay, profound hypotonia, and often the presence of some Rett-syndrome-like features. The association of CDKL5 with neurodevelopmental disorders and its high expression levels in the maturing brain underscore the importance of this kinase for proper brain development. However, our present knowledge of CDKL5 functions is still rather limited. The picture that emerges from the molecular and cellular studies suggests that CDKL5 functions are important for regulating both neuronal morphology through cytoplasmic signaling pathways and activity-dependent gene expression in the nuclear compartment. This paper surveys the current state of CDKL5 research with emphasis on the clinical symptoms associated with mutations in CDKL5, the different mechanisms regulating its functions, and the connected molecular pathways. Finally, based on the available data we speculate that CDKL5 might play a role in neuronal plasticity and we adduce and discuss some possible arguments supporting this hypothesis.
Cyclin-dependent kinase-like 2 (Cdkl2) is a cdc2-related serine/threonine protein kinase that is postnatally expressed in various brain regions, including the cerebral cortex, entorhinal cortex, hippocampus, amygdala, and dorsal thalamus. The extremely high Cdkl2 expression in these regions suggests that it has a role in cognition and emotion. Recent genetic studies indicate that mutations of Cdkl family kinases are associated with neurodevelopmental and neuropsychiatric disorders in humans. To elucidate the physiologic role of Cdkl2, we behaviorally analyzed Cdkl2LacZ/LacZ mice lacking Cdkl2. Cdkl2LacZ/LacZ mice had reduced latencies to enter the dark compartment after electric footshock in an inhibitory avoidance task and attenuated contextual fear responses when exposed to mild training conditions. Hippocampal spatial learning in the Morris water maze was slightly anomalous with mice exhibiting an abnormal swimming pattern. The aversive response in a two-way avoidance task was slightly, but not significantly, enhanced. On the other hand, Cdkl2LacZ/LacZ mice did not exhibit altered sensitivity to aversive stimuli, such as electric footshock and heat, or deficits in the elevated plus maze or rotating rod test. These findings suggest that Cdkl2 is involved in cognitive function and provide in vivo evidence for the function of Cdkl family kinases expressed in terminally differentiated neurons in mice.
active avoidance; Cdkl2; cdc2-related kinase; cognitive function; contextual fear conditioning; inhibitory avoidance; Morris water maze; Rett syndrome
A translocation that disrupted the Netrin G1 gene (NTNG1) was recently reported in a patient with the early seizure variant of Rett syndrome (RTT). The netrin G1 protein (NTNG1) has an important role in the developing central nervous system, particularly in axonal guidance, signalling and NMDA receptor function and was a good candidate gene for RTT. We recruited 115 patients with RTT (females: 25 classic and 84 atypical; 6 males) but no mutation in the MECP2 gene. For those 52 patients with epileptic seizure onset in the first six months of life, CDKL5 mutations were also excluded. We aimed to determine whether mutations in NTNG1 accounted for a significant subset of patients with RTT, particularly those with the early onset seizure variant and other atypical presentations. We sequenced the nine coding exons of NTNG1 and identified four sequence variants, none of which were likely to be pathogenic. Mutations in the NTNG1 gene appear to be a rare cause of RTT but NTNG1 function demands further investigation in relation to the central nervous system pathophysiology of the disorder.
Rett syndrome; Netrin G1; Autism; NMDA receptor
We screened a cohort of 5 male and 20 female patients with a Rett spectrum disorder for mutations in the coding region of FOXG1, previously shown to cause the congenital variant of Rett syndrome. Two de novo mutations were identified. The first was a novel missense mutation, p.Ala193Thr (c.577G>A), in a male patient with congenital Rett syndrome, and the second was the p.Glu154GlyfsX301 (c.460dupG) truncating mutation in a female with classical Rett syndrome, a mutation that was previously reported in an independent patient. The overall rate of FOXG1 mutations in our cohort is 8%. Our findings stress the importance of FOXG1 analysis in male patients with Rett syndrome and in female patients when mutations in the MECP2 and CDKL5 genes have been excluded.
Congenital variant; FOXG1 gene; Male patient; Rett syndrome
The West syndrome (WS) is a characteristic form of epilepsy which usually begins in the first year of life. We describe two female siblings, aged 4 and 2 years, respectively, born from third degree consanguineous parents, with infantile spasms and developmental delay. Epileptic spasms had not a good outcome under antiepileptic drug treatment. Clinical and imaging features were of different severity in both siblings. Routine biochemical tests, metabolic investigations, and chromosomal analysis were normal. We analyzed CDKL5 gene by direct sequences and denaturing high-performance liquid chromatography using Transgenomic WAVE system. Analysis of the CDKL5 gene, which is responsible for female patient with WS, did not show any disease-causing mutation. WS has heterogeneous backgrounds, and may be more than one gene is responsible for its familial forms. In this family, consanguinity is observed in parents, which usually suggests that autosomal recessive inheritance is likely.
Genetics; West syndrome; CDKL5 gene; new genes
Background: Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterised by a wide spectrum of clinical manifestations. Both the classic form and preserved speech variant of Rett syndrome are due to mutations in the MECP2 gene. Several other variants of Rett syndrome have been described. In 1985, Hanefeld described a variant with the early appearance of convulsions. In this variant, the normal perinatal period is soon followed by the appearance of seizures, usually infantile spasms. We have observed two patients with signs of Rett syndrome showing acquired microcephaly and stereotypic midline hand movements. The disease started with generalised convulsions and myoclonic fits at 1.5 months in the first patient and with spasms at 10 days in the other, suggesting a diagnosis of the Hanefeld variant. In these patients, MECP2 point mutations and gross rearrangements were excluded by denaturing high performance liquid chromatography and real time quantitative PCR. The ARX and CDKL5 genes have been associated with West syndrome (infantile spasms, hypsarrhythmia, and mental retardation).
Methods: Based on the clinical overlap between the Hanefeld variant and West syndrome, we analysed ARX and CDKL5 in the two girls.
Results: We found frameshift deletions in CDKL5 in both patients; one in exon 5 (c.163_166delGAAA) and the other in exon 18 (c.2635_2636delCT). CDKL5 was then analysed in 19 classic Rett and 15 preserved speech variant patients, all MECP2 negative, but no mutations were found.
Conclusion: Our results show that CDKL5 is responsible for a rare variant of Rett syndrome characterised by early development of convulsions, usually of the spasm type.
To determine the genetic etiology of the severe early infantile onset syndrome of malignant migrating partial seizures of infancy (MPSI).
Fifteen unrelated children with MPSI were screened for mutations in genes associated with infantile epileptic encephalopathies: SCN1A, CDKL5, STXBP1, PCDH19, and POLG. Microarray studies were performed to identify copy number variations.
One patient had a de novo SCN1A missense mutation p.R862G that affects the voltage sensor segment of SCN1A. A second patient had a de novo 11.06 Mb deletion of chromosome 2q24.2q31.1 encompassing more than 40 genes that included SCN1A. Screening of CDKL5 (13/15 patients), STXBP1 (13/15), PCDH19 (9/11 females), and the 3 common European mutations of POLG (11/15) was negative. Pathogenic copy number variations were not detected in 11/12 cases.
Epilepsies associated with SCN1A mutations range in severity from febrile seizures to severe epileptic encephalopathies including Dravet syndrome and severe infantile multifocal epilepsy. MPSI is now the most severe SCN1A phenotype described to date. While not a common cause of MPSI, SCN1A screening should now be considered in patients with this devastating epileptic encephalopathy.
The study of transcription using genomic tiling arrays has lead to the identification of numerous additional exons. One example is the MECP2 gene on the X chromosome; using 5’RACE and RT-PCR in human tissues and cell lines, we have found more than 70 novel exons (RACEfrags) connecting to at least one annotated exon.. We sequenced all MECP2-connected exons and flanking sequences in 3 groups: 46 patients with the Rett syndrome and without mutations in the currently annotated exons of the MECP2 and CDKL5 genes; 32 patients with the Rett syndrome and identified mutations in the MECP2 gene; 100 control individuals from the same geoethnic group. Approximately 13kb were sequenced per sample, (2.4Mb of DNA resequencing). A total of 75 individuals had novel rare variants (mostly private variants) but no statistically significant difference was found among the 3 groups. These results suggest that variants in the newly discovered exons may not contribute to Rett syndrome. Interestingly however, there are about twice more variants in the novel exons than in the flanking sequences (44 vs. 21 for approximately 1.3 Mb sequenced for each class of sequences, p = 0.0025). Thus the evolutionary forces that shape these novel exons may be different than those of neighboring sequences.
MECP2; Rett syndrome; RACEfrags; SNP; rare variants; positive selection
Rett syndrome is a severe neurodegenerative disorder characterized by acquired microcephaly, communication dysfunction, psychomotor regression, seizures and stereotypical hand movements. Mutations in methyl CpG binding protein 2 (MECP2) are identified in most patients with classic Rett syndrome. Genetic studies in patients with a Rett variant have expanded the spectrum of underlying genetic etiologies. Recently, a deletion encompassing several genes in the long arm of chromosome 14 has been associated with the congenital Rett-syndrome phenotype. Using array-based comparative genomic hybridization, we identified a 3-year-old female with a Rett-like syndrome carrying a de novo single-gene deletion of FOXG1. Her presentation included intellectual disability, epilepsy and a Rett-like phenotype. The variant features included microcephaly at birth and prominent synophrys. Our results confirm that congenital Rett syndrome can be caused by copy-number variation in FOXG1 and expand the clinical phenotypic spectrum of FOXG1 defect in humans.
Rett syndrome; FOXG1; chromosome 14; 14q deletion; array-based comparative genomic hybridization
Rett syndrome is a severe neurodevelopmental disorder in females. Most have mutations in the methyl-CpG-binding protein 2 (MECP2) gene (80-90%). Epilepsy is a significant commonly accompanied feature in Rett syndrome. Our study was aimed at comprehensive analysis of genetic and clinical features in Rett syndrome patients, especially in regards to epileptic features.
Materials and Methods
We retrospectively reviewed 20 patients who were diagnosed with MECP2 mutations at Severance Children's Hospital between January 1995 and July 2010. All patients met clinical criteria for Rett syndrome. Evaluations included clinical features, epilepsy classification, electroencephalography analysis, and treatment of seizures.
Ages ranged from 3.6 to 14.3 years (7.7±2.6). Fourteen different types of MECP2 mutations were found, including a novel in-frame mutation (1153-1188 del36). Fourteen of these patients (70.0%) had epilepsy, and the average age of seizure onset was 3.0±1.8 years. Epilepsy was diverse, including partial seizure in four patients (28.5%), secondarily generalized seizure in six (42.8%), generalized tonic seizure in two (14.3%), Lennox-Gastaut syndrome in one (7.1%), and myoclonic status in non-progressive encephalopathy in one (7.1%). Motor functions were delayed so that only 10 patients (50.0%) were able to walk independently: five (35.8%) in the epilepsy group and five (83.3%) in the non-epilepsy group. Average developmental scale was 33.5±32.8 in the epilepsy group and 44.4±21.2 in the non-epilepsy group. A clear genotype-phenotype correlation was not found.
There is a tendency for more serious motor impairment and cognitive deterioration in Rett syndrome patients with epilepsy.
Rett syndrome; epilepsy; MECP2 mutation
Methyl CpG binding protein-2 (MeCP2) is an essential epigenetic regulator in human brain development. Rett syndrome, the primary disorder caused by mutations in the X-linked MECP2 gene, is characterized by a period of cognitive decline and development of hand stereotypies and seizures following an apparently normal early infancy. In addition, MECP2 mutations and duplications are observed in a spectrum of neurodevelopmental disorders, including severe neonatal encephalopathy, X-linked mental retardation, and autism, implicating MeCP2 as an essential regulator of postnatal brain development. In this review, we compare the mutation types and inheritance patterns of the human disorders associated with MECP2. In addition, we summarize the current understanding of MeCP2 as a central epigenetic regulator of activity-dependent synaptic maturation. As MeCP2 occupies a central role in the pathogenesis of multiple neurodevelopmental disorders, continued investigation into MeCP2 function and regulatory pathways may show promise for developing broad-spectrum therapies.
Rett syndrome; Angelman’s syndrome; Autism; Epigenetic; Neurodevelopmental
Multiplex ligation-dependent Probe Amplification (MLPA) has become available for the detection of a large deletion on the MECP2 gene allowing genetic confirmation of previously unconfirmed cases of clinical Rett syndrome. This study describes the phenotype of those with a large deletion and compares with those with other pathogenic MECP2 mutations. Individuals were ascertained from the Australian Rett Syndrome and InterRett databases with data sourced from family and clinician questionnaires, and two case studies were constructed from the longitudinal Australian data. Regression and survival analysis were used to compare severity and age of onset of symptoms in those with and without a large deletion. Data were available for 974 individuals including 51 with a large deletion and ages ranged from 1 year 4 months to 49 years (median 9 years). Those with a large deletion were more severely affected than those with other mutation types. Specifically, individuals with large deletions were less likely to have learned to walk (OR 0.42, 95% CI: 0.22–0.79, P=0.007) and to be currently walking (OR 0.53, 95% CI: 0.26–1.10, P=0.089), and were at higher odds of being in the most severe category of gross motor function (OR 1.84, 95% CI: 0.98–3.48, P=0.057) and epilepsy (OR 2.72, 95% CI: 1.38–5.37, P=0.004). They also developed epilepsy, scoliosis, hand stereotypies and abnormal breathing patterns at an earlier age. We have described the disorder profile associated with a large deletion from the largest sample to date and have found that the phenotype is severe with motor skills particularly affected.
rett syndrome; large deletion; phenotype
Rett syndrome is a neurodevelopmental and autistic disease caused by mutations of Methyl-CpG-binding protein 2 (MECP2) gene. MeCP2 protein is mainly expressed in neurons and binds to methylated gene promoters to suppress their expression, indicating that Rett syndrome is caused by the deregulation of target genes in neurons. However, it is likely that there are more unidentified neuronal MeCP2-targets associated with the neurological features of RTT.
Using a genome-microarray approach, we found 22 genomic regions that contain sites potentially regulated by MeCP2 based on the features of MeCP2 binding, DNA methylation, and repressive histone modification in human cell lines. Within these regions, Chromatin immunoprecipitation (ChIP) analysis revealed that MeCP2 binds to the upstream regions of the protocadherin genes PCDHB1 and PCDH7 in human neuroblastoma SH-SY5Y cells. PCDHB1 and PCDH7 promoter activities were down-regulated by MeCP2, but not by MBD-deleted MeCP2. These gene expression were up-regulated following MeCP2 reduction with siRNA in SH-SY5Y cells and in the brains of Mecp2-null mice. Furthermore, PCDHB1 was up-regulated in postmortem brains from Rett syndrome patients.
We identified MeCP2 target genes that encode neuronal adhesion molecules using ChIP-on-BAC array approach. Since these protocadherin genes are generally essential for brain development, aberrant regulation of these molecules may contribute to the pathogenesis of the neurological features observed in Rett syndrome.
Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.
Two genes, cyclin-dependent kinase like 3 (cdkl3) and cytochrome c oxidase subunit (cox15), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15. Moreover, flow cytometric analysis indicated that cells with an insert of cdkl3 were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of cox15 was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than cdkl3. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either cdkl3 or cox15.
These results are consistent with previous studies on the functionalities of cdkl3 and cox15. For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to cox15 expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.
Rett syndrome (RTT) is a severe neurodevelopmental disease that affects approximately 1 in 10,000 live female births and is often caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MECP2). Mutations in loci other than MECP2 have also been found in individuals that have been labeled as atypical RTT. Among them, a mutation in the gene forkhead box G1 (FOXG1) has been involved in the molecular aetiology of the congenital variant of RTT. The FOXG1 gene encodes a winged-helix transcriptional repressor essential for the development of the ventral telencephalon in embryonic forebrain. Later, FOXG1 continues to be expressed in neurogenetic zones of the postnatal brain. Although RTT affects quasi-exclusively girls, FOXG1 mutations have also been identified in male patients. As far as we know, about 12 point mutations and 13 cases with FOXG1 molecular abnormalities (including translocation, duplication and large deletion on the chromosome 14q12) have been described in the literature. Affected individuals with FOXG1 mutations have shown dysmorphic features and Rett-like clinical course, including normal perinatal period, postnatal microcephaly, seizures and severe mental retardation. Interestingly, the existing animal models of FOXG1 deficiency showed similar phenotype, suggesting that animal models may be a fascinating model to understand this human disease. Here, we describe the impacts of FOXG1 mutations and their associated phenotypes in human and mouse models.
Chromosome 14; Clinical features; Congenital variant; Encephalopathy; FOXG1; Human; Microcephaly; Molecular basis of disease; Mouse; Rett syndrome
MECP2 mutations are identifiable in ∼80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.
Gross gene deletion; MECP2; Rett syndrome
Rett syndrome (RTT) is a neurodevelopmental disorder primarily seen in females, most with a mutation in MECP2. Epilepsy has been reported in 50%–80%. Previous reports were based on small sample sizes or parent-completed questionnaires, or failed to consider the impact of specific MECP2 mutations.
The Rare Disease Consortium Research Network for RTT is an NIH-funded project to characterize the clinical spectrum and natural history of RTT in advance of clinical trials. Evaluations include clinical status (classic vs atypical RTT), MECP2 mutations, clinical severity, and presence, frequency, and treatment of seizures.
Enrollment as of June 2008 is 602; 528 (88%) meet clinical criteria for classic RTT. Of these, 493 (93%) have MECP2 mutations. Age range was 8 months to 64 years. A total of 360 (60%) were reported to have seizures, including 315 (60%) classic and 45 (61%) atypical RTT. Physician assessment of the 602 indicated that 48% had seizures. There was no significant difference in seizure occurrence by race/ethnicity. A significant age impact for seizures was seen and seizures were infrequent before age 2 years. MECP2 mutations most frequently associated with epilepsy were T158M (74%) and R106W (78%), and less frequently R255X and R306C (both 49%). Individuals with seizures had greater overall clinical severity, and greater impairment of ambulation, hand use, and communication.
Seizures are common in Rett syndrome, have an age-related onset and occurrence, vary by mutation, and are associated with greater clinical severity. This information represents a key consideration for designing clinical trials.
= antiepileptic drug;
= Clinical Severity Scale;
= Motor-Behavioral Assessment;
= Rett syndrome;
= vagus nerve stimulator.
Rett syndrome (RTT) is an X-linked postnatal neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2) and one of the leading causes of mental retardation in females. RTT is characterized by psychomotor retardation, purposeless hand movements, autistic-like behavior and abnormal gait. We studied the effects of environmental enrichment (EE) on the phenotypic manifestations of a RTT mouse model that lacks MeCP2 (Mecp2−/y).
We found that EE delayed and attenuated some neurological alterations presented by Mecp2−/y mice and prevented the development of motor discoordination and anxiety-related abnormalities. To define the molecular correlate of this beneficial effect of EE, we analyzed the expression of several synaptic marker genes whose expression is increased by EE in several mouse models.
We found that EE induced downregulation of several synaptic markers, suggesting that the partial prevention of RTT-associated phenotypes is achieved through a non-conventional transcriptional program.