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Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry+/EGFP+ transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20–35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.

Rett Syndrome (RTT) is an X-linked dominant neurodevelopmental disorder which specifically affecting females. Majority of the cases of RTT is caused because of mutation in methyl-CpG-binding protein (MECP2) gene; besides few cases are caused because of mutation in CDKL5 and FOXG1 gene as well. Since the RTT is a severe disorder and MECP2 and CDKL5 are X linked disorder, therefore, only females are affected. Since, males are hemizygous for X chromosome, therefore, males could not survive with these mutations. In contrast, RTT in males is because of mutations in FOXG1 gene, an autosomal gene. Microcephaly is the prominent phenotype among Rett patients characterized by progressive loss of intellectual functioning, development of stereotypic hand movement, seizures etc. To study the disease pathogenesis, live patients’ neurons are the best suited model system, however, getting patients live brain cells are impossible from an affected individual and also ethically not permissible. Therefore, development of a model system to recapitulate the phenotype of the syndrome with same genome as that of patient is essential. This can be done by using induced pluripotent stem cells (iPSCs) techniques. The main advantage of iPSCs is that it can be used to generate any type of brain cells, even a miniature brain organoid called cerebral organoid can also be generated which can be used to study cross talk between genes and their effects on different brain tissues. The first iPSCs model for Rett was developed by Ellis group in 2009. Subsequently, various researchers successfully generated Rett-iPSCs to evaluate different mutations pertaining to Rett phenotypes. The neurons differentiated from patient-specific iPSCs exhibits similar phenotypes as that of the patients, like reduced soma and nuclear size, decreased dendritic spine density, fewer synapse, altered calcium signaling, etc. It was demonstrated through iPSCs that there is overexpression of GABAergic gene products with MECP2 mutant iPSC-derived neurons while reduced functional synaptic contact and impaired neuronal activity were demonstrated in CDKL5 mutant iPSCs-derived neurons. Moreover, iPSCs-derived neurons from FOXG1-mutated patients showed increased level of GRID1 mRNA levels that regulate synaptic differentiation and thus have altered effect on normal development. Besides the disease pathogenesis study, efforts are also on for cellular therapy as well as evaluating different drug candidates. With the help of latest gene editing technique CRISPR/Cas9, various groups are trying to restore the functionality of MECP2 gene which can lead to a promising way toward treatment for Rett. This technique of iPSCs will be helpful not only for studying RTT in depth but also can pave a path toward drug development to ease the quality of life of the affected children.

The Forkhead transcription factor FOXG1 is a prerequisite for telencephalon development in mammals and is an essential factor controlling expansion of the dorsal telencephalon by promoting neuron and interneuron production. Heterozygous FOXG1 gene mutations cause FOXG1 syndrome characterized by severe intellectual disability, motor delay, dyskinetic movements and epilepsy. Neuroimaging studies in patients disclose constant features including microcephaly, corpus callosum dysgenesis and delayed myelination. Currently, investigative research on the underlying pathophysiology relies on mouse models only and indicates that de-repression of FOXG1 target genes may cause premature neuronal differentiation at the expense of the progenitor pool, patterning and migration defects with impaired formation of cortico-cortical projections. It remains an open question to which extent this recapitulates the neurodevelopmental pathophysiology in FOXG1-haploinsufficient patients. To close this gap, we performed neuropathological analyses in two foetal cases with FOXG1 premature stop codon mutations interrupted during the third trimester of the pregnancy for microcephaly and corpus callosum dysgenesis. In these foetuses, we observed cortical lamination defects and decreased neuronal density mainly affecting layers II, III and V that normally give rise to cortico-cortical and inter-hemispheric axonal projections. GABAergic interneurons were also reduced in number in the cortical plate and persisting germinative zones. Additionally, we observed more numerous PDGFRα-positive oligodendrocyte precursor cells and fewer Olig2-positive pre-oligodendrocytes compared to age-matched control brains, arguing for delayed production and differentiation of oligodendrocyte lineage leading to delayed myelination. These findings provide key insights into the human pathophysiology of FOXG1 syndrome.

FOXG1 syndrome is a neurodevelopmental disorder caused by mutations in the FOXG1 gene. Here, an induced pluripotent stem cell (iPSC) line was generated from human dermal fibroblasts of an individual with the c.490dupG (p.Glu154fs) mutation in the FOXG1 gene. Fibroblasts were reprogrammed using non-integrating episomal plasmids and pluripotency marker expression was confirmed by both immunocytochemistry and quantitative PCR in the resultant iPSC line. There were no karyotypic abnormalities and the cell line successfully differentiated into all three germ layers. This cell line may prove useful in the study of the pathogenic mechanisms that underpin FOXG1 syndrome.

Rett syndrome (RTT) is a rare neurological disorder with a range of clinical manifestations. Mutations in Methyl CpG binding protein 2 (MECP2) gene are found in >95% of typical form and 50–70% of atypical form of Rett syndrome. Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) and Forkhead box G1 (FOXG1) genes are associated with specific RTT variants. This study was carried out to identify the gene mutations in patients with symptoms related to congenital Rett syndrome. Possible causative variations were identified using whole exome sequencing (WES) in two patients. Two novel pathogenic variants (c.563C > A, p.Ala188Glu and c.653A > G, p.Tyr218Cys) were detected in the FOXG1 gene, associated with congenital Rett syndrome. Moreover, other reported variants were also found in the KIF5A and KMT2D genes. In-silico analysis of FOXG1 variants showed that amino acid changes occurred in the conserved domain among different species that altered the conformation of FoxG1 protein; thereby, disrupting the protein function. Our findings extend the variety of mutations in the FOXG1 gene, which cause FOXG1-related syndrome.

FOXG1-related syndrome is a developmental encephalopathy with a high phenotypic variability. A movement disorder presenting at onset is one of the main features, along with microcephaly and severe psychomotor delay without regression. Specific brain MRI findings facilitate the diagnosis. We report three cases of FOXG1-related syndrome, focusing on clinical onset, brain MRI and evolution over time in order to identify common features despite the three different underlying genotypes (14q12 deletion including the FOXG1 gene, FOXG1 intragenic mutation, 14q12 deletion including PRKD1 and a region regulating FOXG1 expression). In conclusion, we stress the importance of considering genetic syndromes in the differential diagnosis of early-onset movement disorders.

More than one hundred distinct gene hemizygosities are specifically linked to epilepsy, mental retardation, autism, schizophrenia and neuro-degeneration. Radical repair of these gene deficits via genome engineering is hardly feasible. The same applies to therapeutic stimulation of the spared allele by artificial transactivators. Small activating RNAs (saRNAs) offer an alternative, appealing approach. As a proof-of-principle, here we tested this approach on the Rett syndrome-linked, haploinsufficient, Foxg1 brain patterning gene. We selected a set of artificial small activating RNAs (saRNAs) upregulating it in neocortical precursors and their derivatives. Expression of these effectors achieved a robust biological outcome. saRNA-driven activation (RNAa) was limited to neural cells which normally express Foxg1 and did not hide endogenous gene tuning. saRNAs recognized target chromatin through a ncRNA stemming from it. Gene upregulation required Ago1 and was associated to RNApolII enrichment throughout the Foxg1 locus. Finally, saRNA delivery to murine neonatal brain replicated Foxg1-RNAa in vivo.

The cre/loxP system is used routinely to manipulate gene expression in the mouse nervous system. In order to delete genes specifically from the telencephalon, the Foxg1-cre line was created previously by replacing the intron-less Foxg1 coding region with cre, resulting in a Foxg1 heterozygous mouse. As the telencephalon of heterozygous Foxg1 mice was reported to be normal, this genotype often has been used as the control in subsequent analyses. Here we describe substantial disruption of forebrain development of heterozygous mice in the Foxg1-cre line, maintained on the C57BL/6J background. High resolution magnetic resonance microscopy reveals a significant reduction in the volume of the neocortex, hippocampus and striatum. The alteration in the neocortex results, in part, from a decrease in its tangential dimension, although gross patterning of the cortical sheet appears normal. This decrease is observed in three different Foxg1 heterozygous mouse lines, independent of the method of achieving deletion of the Foxg1 gene. Although Foxg1 is not expressed in the diencephalon, three-dimensional magnetic resonance microscopy revealed that thalamic volume in the adult is reduced. In contrast, at postnatal day 4, thalamic volume is normal, suggesting that interactions between cortex and dorsal thalamus postnatally produce the final adult thalamic phenotype. In the Foxg1-cre line maintained on the C57BL/6J background, the radial domain of the cerebral cortex also is disrupted substantially, particularly in supragranular layers. However, neither Foxg1 heterozygous mice of the Foxg1-tet (tetracycline transactivator) line, nor those of the Foxg1-lacZ and Foxg1-cre lines maintained on a mixed background, displayed a reduced cortical thickness. Thus Cre recombinase contributes to the radial phenotype, although only in the context of the congenic C57BL/6J background. These observations highlight an important role for Foxg1 in cortical development, reveal noteworthy complexity in the invocation of specific mechanisms underlying phenotypes expressed following genetic manipulations and stress the importance of including appropriate controls of all genotypes.

Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by the progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, deceleration of head growth, and the development of stereotypic hand movements, occurring after a period of normal development. The classic form of RTT involves mutation in MECP2 while the involvement of CDKL5 and FOXG1 genes has been identified in atypical RTT phenotype. FOXG1 gene encodes for a fork-head box protein G1, a transcription factor acting primarily as transcriptional repressor through DNA binding in the embryonic telencephalon as well as a number of other neurodevelopmental processes. In this report we have described the molecular analysis of FOXG1 gene in Indian patients with Rett syndrome. FOXG1 gene mutation analysis was done in a cohort of 34 MECP2/CDKL5 mutation negative RTT patients. We have identified a novel mutation (p. D263VfsX190) in FOXG1 gene in a patient with congenital variant of Rett syndrome. This mutation resulted into a frameshift, thereby causing an alteration in the reading frames of the entire coding sequence downstream of the mutation. The start position of the frameshift (Asp263) and amino acid towards the carboxyl terminal end of the protein was found to be well conserved across species using multiple sequence alignment. Since the mutation is located at forkhead binding domain, the resultant mutation disrupts the secondary structure of the protein making it non-functional. This is the first report from India showing mutation in FOXG1 gene in Rett syndrome.

The Forkhead box G1 (FOXG1) gene encodes a transcriptional repressor essential for early development of the telencephalon. Intragenic mutations and gene deletions leading to haploinsufficiency cause the congenital variant of Rett syndrome. We here describe Rett syndrome-like patients, three of them carrying a balanced translocation with breakpoint in the chromosome 14q12 region, and one patient having a 14q12 microdeletion excluding the FOXG1 gene. The hypothesis of long-range FOXG1-regulatory elements in this region was supported by our finding of reduced FOXG1 mRNA and protein levels in platelets and skin fibroblasts from these cases. Given that FOXG1 is not only expressed in brain but also in platelets, we have studied platelet morphology in these patients and two additional patients with FOXG1 mutations. Electron microscopy of their platelets showed some enlarged, rounder platelets with often abnormal alpha, and fewer dense granules. Platelet function studies were possible in one 14q12 translocation patient with a prolonged Ivy bleeding time and a patient with a heterozygous FOXG1 c.1248C>G mutation (p.Tyr416X). Both have a prolonged PFA-100 occlusion time with collagen and epinephrine and reduced aggregation responses to low dose of ADP and epinephrine. Dense granule ATP secretion was normal for strong agonists but absent for epinephrine. In conclusion, our study shows that by using platelets functional evidence of cis-regulatory elements in the 14q12 region result in reduced FOXG1 levels in patients' platelets having translocations or deletions in that region. These platelet functional abnormalities deserve further investigation regarding a non-transcriptional regulatory role for FOXG1 in these anucleated cells.European Journal of Human Genetics advance online publication, 1 May 2013; doi:10.1038/ejhg.2013.86. ispartof: European Journal of Human Genetics vol:21 issue:12 pages:1349-1355 ispartof: location:England status: published

The Forkhead box G1 (FOXG1) gene has been implicated in severe Rett-like phenotypes. It encodes the Forkhead box protein G1, a winged-helix transcriptional repressor critical for forebrain development. Recently, the core FOXG1 syndrome was defined as postnatal microcephaly, severe mental retardation, absent language, dyskinesia, and dysgenesis of the corpus callosum. We present seven additional patients with a severe Rett-like neurodevelopment disorder associated with de novo FOXG1 point mutations (two cases) or 14q12 deletions (five cases). We expand the mutational spectrum in patients with FOXG1-related encephalopathies and precise the core FOXG1 syndrome phenotype. Dysgenesis of the corpus callosum and dyskinesia are not always present in FOXG1-mutated patients. We believe that the FOXG1 gene should be considered in severely mentally retarded patients (no speech-language) with severe acquired microcephaly (−4 to−6 SD) and few clinical features suggestive of Rett syndrome. Interestingly enough, three 14q12 deletions that do not include the FOXG1 gene are associated with phenotypes very reminiscent to that of FOXG1-mutation-positive patients. We physically mapped a putative long-range FOXG1-regulatory element in a 0.43 Mb DNA segment encompassing the PRKD1 locus. In fibroblast cells, a cis-acting regulatory sequence located more than 0.6 Mb away from FOXG1 acts as a silencer at the transcriptional level. These data are important for clinicians and for molecular biologists involved in the management of patients with severe encephalopathies compatible with a FOXG1-related phenotype.

Microduplications on chromosome 14q11.2 including the FOXG1 gene have been reported in patients with developmental delay, cognitive impairment with speech delay, and epilepsy.1, 2 Such association has been confirmed subsequently in other patients.3, 4 However, Amor et al5 have found an ∼88 kb duplication at 14q12, encompassing the FOXG1 and C14orf23 genes in a father-son pair with isolated hemifacial microsomia. Neither the son nor the father exhibited mental retardation or epilepsy. They also identified an ∼3 Mb duplication of the 14q12 region, including FOXG1, in a child enrolled as a control subject in the CHOP CNV database6 and questioned the pathogenicity of FOXG1 duplication. We believe it is important to highlight that the clinical phenotypes observed in the seven patients in the original description of the syndrome include a relatively wide spectrum of neurodevelopmental abnormalities, ranging from mild to severe intellectual disability and variable presence of epilepsy (in four out of the seven patients).1 Thus, it is not surprising that subjects at the mildest end of the spectrum may present with few or no clinically evident manifestations of the disease. Moreover, the duplicated copy of FOXG1 reported by Amor et al5 is small and may be devoid of its distant regulatory elements, which may explain the lack of neurocognitive phenotype. In support of this notion, two other FOX genes, FOXF1 and FOXL2, encoding for the evolutionarily conserved family of transcription factors with a central role in development have been shown recently to be upregulated by non-coding copy-number variants mapping over 250 kb 5′ from these genes.7, 8 Of note, FOXG1 expression is restricted to the brain, and thus more likely to be finely regulated by such distant enhancer(s) in a tissue-specific manner. With regard to the individual in the CHOP CNV database with a FOXG1 duplication, we agree with the authors' suggestion that the CHOP subject with the duplication of FOXG1 may have not manifested yet the neurodevelopmental abnormalities. On the basis of the well-established causative role of genomic deletions and point mutations of FOXG1 in determining a Rett-like phenotype9, 10 and the studies generated in animal models,11 the evidence of FOXG1 as a dosage sensitive gene is compelling. Thus, we believe microduplications involving FOXG1 should not be considered of questionable pathogenicity but rather highly likely to be pathogenic, albeit associated with a wide spectrum of abnormalities, which is commonly observed with other microduplication syndromes.12

Item does not contain fulltext Mutations in the Forkhead box G1 (FOXG1) gene, a brain specific transcriptional factor, are responsible for the congenital variant of Rett syndrome. Until now FOXG1 point mutations have been reported in 12 Rett patients. Recently seven additional patients have been reported with a quite homogeneous severe phenotype designated as the FOXG1 syndrome. Here we describe two unrelated patients with a de novo FOXG1 point mutation, p.Gln46X and p.Tyr400X, respectively, having a milder phenotype and sharing a distinctive facial appearance. Although FoxG1 action depends critically on its binding to chromatin, very little is known about the dynamics of this process. Using fluorescence recovery after photobleaching, we showed that most of the GFP-FoxG1 fusion protein associates reversibly to chromatin whereas the remaining fraction is bound irreversibly. Furthermore, we showed that the two pathologic derivatives of FoxG1 described in this paper present a dramatic alteration in chromatin affinity and irreversibly bound fraction in comparison with Ser323fsX325 mutant (associated with a severe phenotype) and wild type Foxg1 protein. Our observations suggest that alterations in the kinetics of FoxG1 binding to chromatin might contribute to the pathological effects of FOXG1 mutations.

Null mutation of the Foxg1 gene causes hypoplasia of the mouse telencephalon and loss of ventral telencephalic structures. We show that a crucial early requirement for Foxg1 is in the induction of ventral cell fate in the telencephalon. To study later proliferative defects, we have adapted an iododeoxyuridine and bromodeoxyuridine double labeling protocol for use in the developing embryo, which allows estimation of cell cycle kinetics in a single specimen. This technique is used to demonstrate that the cell cycle is prematurely lengthened in the Foxg1-null telencephalon. These defects are first apparent at embryonic day 10.5 (E10.5) and are most severe in the rostral telencephalon. We show that apoptosis is also reduced in the same rostral domain. These defects correspond temporally and spatially with a dramatic reduction in expression of the potent signaling molecule Fgf8. We also show that in the absence of Foxg1 an excess of neurons is produced from E11.5, depleting the progenitor pool and limiting the growth of the Foxg1−/− telencephalon. The increase in neurogenic division coincides with an increase in BMP signaling, as detected by immunohistochemistry for phosphorylated smad-1, -5, and -8. This study reinforces Foxg1's position as a major regulator of telencephalic neurogenesis and supports the idea that Foxg1 controls precursor proliferation via regulation of Fgf signaling and differentiation via regulation of Bmp signaling.

The FOXG1 syndrome is emerging as a relative new entity in paediatric neurology. We report a boy with acquired microcephaly, mental retardation and a thin genu of the corpus callosum. The combination of these findings led to mutation analysis of FOXG1 . The patient was found to be heterozygous for a novel mutation in FOXG1 , c.506dup (p.Lys170GInfsX285), which occurred de novo . This frameshift mutation disturbs the three functional domains of the FOXG1 gene. Hypo- or agenesis of the anterior corpus callosum in combination with acquired microcephaly and neurologic impairment can be an important clue for identifying patients with a mutation in FOXG1 .