Juliette Nectoux

Key clinical features to identify girls with CDKL5 mutations

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause infantile spasms as well as Rett syndrome (RTT)-like phenotype. To date, less than 25 different mutations have been reported. So far, there are still little data on the key clinical diagnosis criteria and on the natural history of CDKL5-associated encephalopathy. We screened the entire coding region of CDKL5 for mutations in 183 females with encephalopathy with early seizures by denaturing high liquid performance chromatography and direct sequencing, and we identified in 20 unrelated girls, 18 different mutations including 7 novel mutations. These mutations were identified in eight patients with encephalopathy with RTT-like features, five with infantile spasms and seven with encephalopathy with refractory epilepsy. Early epilepsy with normal interictal EEG and severe hypotonia are the key clinical features in identifying patients likely to have CDKL5 mutations. Our study also indicates that these patients clearly exhibit some RTT features such as deceleration of head growth, stereotypies and hand apraxia and that these RTT features become more evident in older and ambulatory patients. However, some RTT signs are clearly absent such as the so called RTT disease profile (period of nearly normal development followed by regression with loss of acquired fine finger skill in early childhood and characteristic intensive eye communication) and the characteristic evolution of the RTT electroencephalogram. Interestingly, in addition to the overall stereotypical symptomatology (age of onset and evolution of the disease) resulting from CDKL5 mutations, atypical forms of CDKL5-related conditions have also been observed. Our data suggest that phenotypic heterogeneity does not correlate with the nature or the position of the mutations or with the pattern of X-chromosome inactivation, but most probably with the functional transcriptional and/or translational consequences of CDKL5 mutations. In conclusion, our report show that search for mutations in CDKL5 is indicated in girls with early onset of a severe intractable seizure disorder or infantile spasms with severe hypotonia, and in girls with RTT-like phenotype and early onset seizures, though, in our cohort, mutations in CDKL5 account for about 10% of the girls affected by these disorders.

Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant

The Forkhead box G1 (FOXG1) is a transcription factor that is critical for forebrain development, where it promotes progenitor proliferation and suppresses premature neurogenesis. Recently, the FOXG1 gene was implicated in the molecular aetiology of the congenital variant of Rett syndrome. So far, 15 FOXG1 molecular alterations, including only eight point mutations, have been reported. We screened the FOXG1 gene in a cohort of 206 MECP2 and CDKL5 mutation negative patients (136 females and 70 males) with severe encephalopathy and microcephaly. The screening was negative in all males, but two de novo mutations (c.1248C>G, p.Y416X and c.460_461dupG, p.E154GfsX300) were identified in two unrelated girls. Both patients showed neurological symptoms from the neonatal period with poor reactivity, hypotonia, and severe microcephaly. During the first year of life, both patients had feeding difficulties and made slow developmental progress. At 5xa0years old, the girls were significantly neurologically impaired with gross hypotonia, no language, convergent strabismus, and no voluntary hand use. Moreover, they presented a combination of jerky movements, hand-mouthing, and hand-washing stereotypies. Hence, FOXG1 mutation patients demonstrate severe encephalopathy compatible with the congenital variant, as well as additional features such as absent eye contact, inconsolable crying during the perinatal period, and delayed myelination with thin to hypoplastic corpus callosum. Although the overall frequency of mutations in FOXG1 in females with severe mental retardation and microcephaly appears to be low (1.5%), our findings suggest the requirement to investigate both point mutations and gene dosage in the FOXG1 gene in patients with severe encephalopathy with microcephaly and some Rett-like features.

Epileptic encephalopathy in a girl with an interstitial deletion of Xp22 comprising promoter and exon 1 of the CDKL5 gene

We report a 2‐year‐old girl with early onset seizures variant of Rett syndrome with a deletion at Xp22 detected by multiplex ligation‐dependent probe amplification (MLPA) technique. This patient presented with tonic seizures at 7 days of life. Subsequently, she developed infantile spasms at three months and finally refractory myoclonic epilepsy. She demonstrated severe encephalopathy with hypotonia, deceleration of head growth, with eye gaze but limited eye pursuit, no language, limited hand use, and intermittent hand stereotypies. This combination of clinical features, suggestive of early onset variant of Rett syndrome led us to screen the CDKL5 gene. In a first step, screening of the whole coding sequence of the CDKL5 gene revealed no point mutations. In a second step, we searched gross rearrangements by MLPA and identified a microdeletion affecting both the promoter and exon 1 in CDKL5. Subsequent analysis on a Nimblegen HD2 microarray confirmed a deletion of approximately 300 kb at Xp22, including the BEND2, SCML2, and CDKL5 genes. In conclusion, our report suggests that searching for large rearrangements in CDKL5 should be considered in girls with early onset seizures and Rett‐like features.

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.

Cell cloning‐based transcriptome analysis in Rett patients: relevance to the pathogenesis of Rett syndrome of new human MeCP2 target genes

More than 90% of Rett syndrome (RTT) patients have heterozygous mutations in the X‐linked methyl‐CpG binding protein 2 (MECP2) gene that encodes the methyl‐CpG‐binding protein 2, a transcriptional modulator. Because MECP2 is subjected to X chromosome inactivation (XCI), girls with RTT either express the wild‐type or mutant allele in each individual cell. To test the consequences of MECP2 mutations resulting from a genome‐wide transcriptional dysregulation and to identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we carried out gene expression profiling of clonal populations derived from fibroblast primary cultures expressing exclusively either the wild‐type or the mutant MECP2 allele. Clonal cultures were obtained from skin biopsy of three RTT patients carrying either a non‐sense or a frameshift MECP2 mutation. For each patient, gene expression profiles of wild‐type and mutant clones were compared by oligonucleotide expression microarray analysis. Firstly, clustering analysis classified the RTT patients according to their genetic background and MECP2 mutation. Secondly, expression profiling by microarray analysis and quantitative RT‐PCR indicated four up‐regulated genes and five down‐regulated genes significantly dysregulated in all our statistical analysis, including excellent potential candidate genes for the understanding of the pathophysiology of this neurodevelopmental disease. Thirdly, chromatin immunoprecipitation analysis confirmed MeCP2 binding to respective CpG islands in three out of four up‐regulated candidate genes and sequencing of bisulphite‐converted DNA indicated that MeCP2 preferentially binds to methylated‐DNA sequences. Most importantly, the finding that at least two of these genes (BMCC1 and RNF182) were shown to be involved in cell survival and/or apoptosis may suggest that impaired MeCP2 function could alter the survival of neurons thus compromising brain function without inducing cell death.

Altered microtubule dynamics in Mecp2-deficient astrocytes.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in the gene MECP2 encoding the methyl‐CpG binding protein 2. This genetic disease affects predominantly girls and is characterized by a period of normal development that lasts for 8–18 months, followed by neurologic regression affecting both motor and mental abilities. Previous studies performed on brains from RTT subjects and Mecp2‐deficient mice showed striking changes in neuronal maturation and dendritic arborization. Recently, we showed that expression of stathmin‐like 2 (STMN2) was significantly reduced in fibroblasts from RTT patients, and similar results were obtained in the cerebellum of Mecp2‐deficient mice. Because assembly and dynamics of microtubules are known to be modulated by STMN2, we studied microtubule dynamics in brain cells from Mecp2‐deficient mice. We observed that Mecp2 deficiency affects microtubule dynamics in astrocytes from Mecp2‐deficient mice. Our data reinforce the fact that the loss of Mecp2 in astrocytes may influence the onset and progression of RTT. These results imply that Mecp2 has a stabilizing role in microtubule dynamics and that Mecp2 deficiency, which is associated with STMN2 down‐regulation, could lead to impaired microtubule stability, hence explaining the dendritic abnormalities observed in RTT brains.

MeCP2 deficiency is associated with impaired microtubule stability.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Previous studies performed on Mecp2‐deficient brain showed striking changes in neuronal maturation. We recently showed that MeCP2 deficiency affects microtubule (MT) dynamics in RTT astrocytes. Here, we analyze MT stability in primary fibroblast cultures from patients with RTT syndrome and identify a significant decrease in stability compared to controls. Furthermore, we found that MT stability was reduced both in cells expressing the mutant or the wild‐type allele in RTT fibroblasts, suggesting that mutated cells could damage wild‐type ones through a non‐cell‐autonomous pathway. These results suggest that MeCP2 has a stabilizing role on MT dynamics and that its deficiency could lead to impaired MT stability that may explain in part the dendritic abnormalities observed in RTT brains.

A missense mutation within the fork-head domain of the forkhead box G1 Gene (FOXG1) affects its nuclear localization†

The forkhead box G1 (FOXG1)gene has recently been associated with the congenital variant of Rett syndrome, and so far 17 mutations have been reported. We screened the coding region in 150 patients affected by postnatal microcephaly, and identified two mutations: the c.326C>T (p.P109L) substitution inherited from the healthy father; and the de novo c.730C>T transition, which induces the p.R244C mutation within the DNA‐binding forkhead domain. This latter mutation is carried by an 8‐year‐old girl, who presented a phenotype reminiscent of the congenital variant of Rett syndrome. Immunofluorescence analysis of the wild‐type protein revealed a homogeneous nuclear staining excluding the nucleoli, while the p.R244C mutant showed abnormal nuclear foci in a large proportion of cells, suggesting that its mislocalization may reduce and/or impair target recognition. Interestingly, this missense mutation results in a mislocalization of FoxG1 to specific nuclear foci referred to as nuclear speckles, and affects the cyclin‐dependent kinase inhibitor p21 CDKN1A expression. Because CDKL5, which is involved in the early‐onset variant of Rett syndrome, is also located in these speckles, we suggest that disregulation of the dynamic behaviour of nuclear speckles may functionally link these two proteins, which are both involved in atypical forms of Rett syndrome.

Cell cloning-based transcriptome analysis in cyclin-dependent kinase-like 5 mutation patients with severe epileptic encephalopathy

Mutations in the human CDKL5 gene have been shown to cause infantile spasms, as well as Rett syndrome-like phenotype. Because CDKL5 is subjected to X chromosome inactivation (XCI), individual cells from CDKL5 mutation girls either express the wild-type or mutant allele, likely resulting in different consequences at both the cellular and molecular levels. To identify these consequences, we carried out gene expression profiling on clonal populations derived from primary cultures of three patients fibroblasts either expressing the wild-type or mutant allele. A total of 16 up-regulated and 20 down-regulated genes were identified. The differentially expressed gene products, mostly involved in differentiation and oxidative stress may be related to a mechanism underlying mental retardation and epilepsy. Among these, the apoptosis signal-regulated kinase MAP3K5 expression was found to be altered in non-neuronal, but also in neuronal CDKL5-deficient cells. Due to the fact that MAP3K5 activates MAP kinase pathway, which mediates signals leading to both differentiation and survival in neuronal cells, we suggest that a CDKL5 deficit may induce changes in synaptic plasticity in the patients brain.

Somatic mosaicism for a FOXG1 mutation: diagnostic implication.

To the Editor : In 2005, Shoichet et al. reported a girl exhibiting severe cognitive disability with a balanced de novo translocation that disrupts the winged-helix transcription factor forkhead box G1 (FOXG1 ) gene (1). Later, three 14q12 interstitial deletions including FOXG1 were identified in patients with intellectual disability, epilepsy, microcephaly, and facial dysmorphism (2). Finally, FOXG1 -null mutations were reported in the congenital variant of Rett syndrome (RTT) in two unrelated girls (3). Up to now, 12 FOXG1 point mutations have been identified in patients suffering from typical and atypical forms of RTT (2). In all cases, FOXG1 point mutations were or appeared to be de novo. Here, we report a 2-year-old boy with a novel FOXG1 mutation, transmitted by an unaffected mother who was found to show somatic mosaicism for the mutation. Familial occurrence and germline mosaicism for FOXG1 mutation have been reported only once, in a case of duplication (4). This patient was born at term after uneventful pregnancy. His psychomotor development was delayed from the second month of life. He held his head at the age of 1 year but has never been able to sit unaided or walk. He never acquired spoken language and had poor eye contact. He developed infantile spasms at 4 month old without hypsarrhythmia. There was no dysmorphism. Clinical and physical examinations at the age of 1 year revealed growth retardation, severe axial and peripheral hypotonia, microcephaly [occipitofrontal circumference (OFC) 41.5 cm, −4 standard deviation (SD)], severe generalized dyskinesia and permanent hand to mouth stereotypies. A brain magnetic resonance imaging (MRI) study showed cerebral atrophy. Conventional cytogenetic investigations were normal. After standard DNA extraction and polymerase chain reaction (PCR) amplification, direct sequencing of FOXG1 coding sequences in patient’s DNA identified a novel heterozygous mutation in coding exon 1. The c.974_975insA mutation (p.Ser326Glufs*129) causes the loss of the JARID1B binding domain. This mutation was not observed among 200 control chromosomes. The mutation was absent from the father’s DNA. However, direct sequencing of the mother’s DNA suggested the presence of a somatic mosaicism (Fig. 1), because a very small amount of the mutated residue was detectable at different positions following c.974 position, and the signal was too weak to be considered as a heterozygous mutation. In order to determine the level of mosaicism, we developed a real-time allele-specific PCR (AS-PCR) assay for this mutation. The mutant-specific forward primer was 5′-CCCGCGCCAGCAGCACTTTA-3′ and the reverse primer was 5′-ACGCTCAGGCCGTTGG CGGT-3′. Quantitative detection of this mutant was developed using the SYBR Green PCR Master Mix. The melting curve analysis revealed that the mutantspecific amplicon melted at 85.59◦C. The standard curve for the mutant allele was generated by a serial dilution of the proband’s DNA with the wild-type DNA on an ABI Prism 7000 sequence detection system (R2 = 0.98). Levels of the mutant allele in the mother sample were calculated based on the value of delta computed tomography and the standard curve. Results showed that 35.6% of leukocytes harboured the mutation. In order to confirm our results, mosaicism was quantified in another tissue. A skin biopsy was obtained from the mother and primary cultures of fibroblasts were performed. DNA was extracted from fibroblast culture, and similar result was obtained although the level of mosaicism was lower in fibroblasts than in peripheral blood cells. We estimated the level of the mosaicism at 9.7% in mother’s fibroblasts. Mosaicism has been documented for chromosomal abnormalities, mitochondrial mutations, triplet repeats, and in growing number of dominant and recessive X-linked gene disorders (5). Because a proportion of cells carry the mutation not only in blood but also in other tissues deriving from other cell lineages, it must be assumed that the mutation must have occurred early during embryogenesis in a cell that contributed to both germline and somatic tissues. We show for the first time to our knowledge that somatic mosaicism for FOXG1 mutations in individuals is not infrequent. This report has important consequences for genetic counselling. The occurrence of somatic and germ cell mosaicism represents a matter of concern to the genetic counsellor, because it causes uncertainty about the recurrence risk in parents who appear to be non-carriers using classical approaches.