FOXG1 Syndrome was first identified in 2005 and has been closely associated with Rett Syndrome. However, with access to genetic testing an increasing number of patients have been identified that do not fulfill Rett Syndrome clinical criteria. Utilizing genetic surveys of large, severe neuro-developmental disease (NDD) cohorts, we provide the first prevalence estimates of FOXG1 Syndrome independent of Rett Syndrome clinical criteria.

Heterozygous mutations in the FOXG1 gene manifest as FOXG1 syndrome, a severe neurodevelopmental disorder characterized by structural brain anomalies, including agenesis of the corpus callosum, hippocampal reduction, and myelination delays. Despite the well-defined genetic basis of FOXG1 syndrome, therapeutic interventions targeting the underlying cause of the disorder are nonexistent. In this study, we explore the therapeutic potential of adeno-associated virus 9 (AAV9) mediated delivery of the FOXG1 gene. Remarkably, intracerebroventricular injection of AAV9-FOXG1 to Foxg1 heterozygous mouse model at the postnatal stage rescues a wide range of brain pathologies. This includes the amelioration of corpus callosum deficiencies, the restoration of dentate gyrus morphology in the hippocampus, the normalization of oligodendrocyte lineage cell numbers, and the rectification of myelination anomalies. Our findings highlight the efficacy of AAV9-based gene therapy as a viable treatment strategy for FOXG1 syndrome and potentially other neurodevelopmental disorders with similar brain malformations, asserting its therapeutic relevance in postnatal stages.

Background: We refine the clinical spectrum of FOXG1 syndrome and expand genotype-phenotype correlations through evaluation of 122 individuals enrolled in an international patient registry.

Methods: The FOXG1 syndrome online patient registry allows for remote collection of caregiver-reported outcomes. Inclusion required documentation of a (likely) pathogenic variant in FOXG1. Caregivers were administered a questionnaire to evaluate clinical severity of core features of FOXG1 syndrome. Genotype-phenotype correlations were determined using nonparametric analyses.

FOXG1 syndrome is a rare neurodevelopmental disorder associated with severe cognitive dysfunction, autistic behavior, and early-onset hyperkinetic movement disorders. Patients have also been reported to experience sleep disturbances. However, these findings are mainly based on subjective caregivers' reports, and limited by small case numbers. Moreover, no studies using objective evaluation tools, such as actigraphy, have been reported.

FOXG1 syndrome (FS, aka a congenital variant of Rett syndrome) is a recently defined rare and devastating neurodevelopmental disorder characterized by various symptoms, including severe intellectual disability, autistic features, involuntary, and continuous jerky movements, feeding problems, sleep disturbances, seizures, irritability, and excessive crying. FS results from mutations in a single allele of the FOXG1 gene, leading to impaired FOXG1 function. Therefore, in establishing mouse models for FS, it is important to test if heterozygous (HET) mutation in the Foxg1 gene, mimicking genotypes of the human FS individuals, also manifests phenotypes similar to their symptoms. We analyzed HET mice with a null mutation allele in a single copy of Foxg1, and found that they show various phenotypes resembling the symptoms of the human FS individuals. These include increased anxiety in the open field as well as impairment in object recognition, motor coordination, and fear learning and contextual and cued fear memory. Our results suggest that Foxg1 HET mice recapitulate at least some symptoms of the human FS individuals.

Abnormalities in GABAergic inhibitory circuits have been implicated in the aetiology of autism spectrum disorder (ASD). ASD is caused by genetic and environmental factors. Several genes have been associated with syndromic forms of ASD, including FOXG1. However, when and how dysregulation of FOXG1 can result in defects in inhibitory circuit development and ASD-like social impairments is unclear. Here, we show that increased or decreased FoxG1 expression in both excitatory and inhibitory neurons results in ASD-related circuit and social behavior deficits in our mouse models. We observe that the second postnatal week is the critical period when regulation of FoxG1 expression is required to prevent subsequent ASD-like social impairments. Transplantation of GABAergic precursor cells prior to this critical period and reduction in GABAergic tone via Gad2 mutation ameliorates and exacerbates circuit functionality and social behavioral defects, respectively. Our results provide mechanistic insight into the developmental timing of inhibitory circuit formation underlying ASD-like phenotypes in mouse models.

Brain development is a highly regulated process that involves the precise spatio-temporal activation of cell signaling cues. Transcription factors play an integral role in this process by relaying information from external signaling cues to the genome. The transcription factor Forkhead box G1 (FOXG1) is expressed in the developing nervous system with a critical role in forebrain development. Altered dosage of FOXG1 due to deletions, duplications, or functional gain- or loss-of-function mutations, leads to a complex array of cellular effects with important consequences for human disease including neurodevelopmental disorders. Here, we review studies in multiple species and cell models where FOXG1 dose is altered. We argue against a linear, symmetrical relationship between FOXG1 dosage states, although FOXG1 levels at the right time and place need to be carefully regulated. Neurodevelopmental disease states caused by mutations in FOXG1 may therefore be regulated through different mechanisms.

Our brain comprises 85 billion nerve cells and just as many so-called glial cells, which work in close contact with the former to guarantee their proper function. All originate from brain stem cells. But what decides when and how many of them become neurons or glial cells? A new study led by the Laboratory of Cerebral Cortex Development of SISSA has shown how the Foxg1 gene, already involved in numerous processes of cerebral development and in rare disorders like Rett and West syndromes, plays a fundamental role in piloting the differentiation of stem cells, guaranteeing that neurons and glial cells are produced in the right quantity and at the right moment. The work, published in Cerebral Cortex and conducted in collaboration with the University of Cambridge and the IRCCS Burlo Garofolo, opens new roads to understanding and treating incurable genetic diseases.

Dosage of key regulators impinge on developmental disorders such as FOXG1 syndrome. Since neither knock-out nor knock-down strategy assures flexible and precise protein abundance control, to study hypomorphic or haploinsufficiency expression remains challenging. We develop a system in human pluripotent stem cells (hPSCs) using CRISPR/Cas9 and SMASh technology, with which we can target endogenous proteins for precise dosage control in hPSCs and at multiple stages of neural differentiation. We also reveal FOXG1 dose-dependently affect the cellular constitution of human brain, with 60% mildly affect GABAergic interneuron development while 30% thresholds the production of MGE derived neurons. Abnormal interneuron differentiation accounts for various neurological defects such as epilepsy or seizures, which stimulates future innovative cures of FOXG1 syndrome. By means of its robustness and easiness, dosage-control of proteins in hPSCs and their derivatives will update the understanding and treatment of additional diseases caused by abnormal protein dosage.

The hallmarks of FOXG1 syndrome, which results from mutations in a single FOXG1 allele, include cortical atrophy and corpus callosum agenesis. However, the etiology for these structural deficits and the role of FOXG1 in cortical projection neurons remain unclear. Here we demonstrate that Foxg1 in pyramidal neurons plays essential roles in establishing cortical layers and the identity and axon trajectory of callosal projection neurons. The neuron-specific actions of Foxg1 are achieved by forming a transcription complex with Rp58. The Foxg1-Rp58 complex directly binds and represses Robo1, Slit3, and Reelingenes, the key regulators of callosal axon guidance and neuronal migration. We also found that inactivation of one Foxg1allele specifically in cortical neurons was sufficient to cause cerebral cortical hypoplasia and corpus callosum agenesis.

A new case series led by Dr. Bahi-Buisson adds 45 additional ‘foxes’ to the medical literature (bringing the total number >130). Importantly, this study increases the range of ages reported in FOXG1 syndrome - 6 of the new cases are 18 years or older, providing valuable information about what life is like for affected adults. As seen in other case series, the majority of causative mutations are ‘frameshift’ variants that remove part of the FOXG1 protein.

The authors compared the clinical features of 83 individuals to determine whether any patterns could be identified based on the types of observed pathogenic variants (frameshift, missense, nonsense).

In general, those individuals with truncating variants in the N-terminal (beginning) domain of FOXG1 had a more severe presentation. Individuals with pathogenic missense variants in an evolutionarily conserved region of the forkhead
DNA-binding domain had milder presentations. This article also reports if and when individuals achieved developmental milestones: 45% were able to sit unassisted at a mean age of 28 months; 15% were able to walk unsupported at a mean age
of 53 months; functional hand use observed in 40%; 21% had some verbal expression (note that age at last follow-up was not consistent, and different for each individual).

Foxg1-RNAa delivered to murine E12.5 neocortical precursors by lentivirus; expression increase ranged from 1.28-2.88 fold increase. Increasing Foxg1expression in proliferating murine neocortical precursors decreased the number of neurons generated.

By delivering the RNAa molecule to specific neuronal populations, the authors provided evidence to suggest that ectopic (or off-target) up-regulation of Foxg1expression would not be significant.

iPSC neurons generated from families with a proband dx with idiopathic ASD and increased head circumference; FOXG1 expression consistently shown to be upregulated in these individuals.

shRNA designed to target and downregulate FOXG1 expression; when ASD-derived iPSC cells treated, FOXG1 expression levels lowered to a level similar to their unaffected controls, with downregulation of GABAergic markers, with restoration to the normal level of GABAergic neuronal differentiation. Evidence also suggest upregulation of FOXG1 in ASD neural cells is an early driving force for proliferation of npcs of GABAergic lineage.

iPSC neurons generated from fibroblasts of two individuals with FOXG1 syndrome, one carrying a truncating variant, and the other with a deletion including the entire FOXG1 gene.

Similar to what is found in iPSC neurons from individuals with CDKL5 or MECP2-related disorders, expression of the gene, GRID1, was significantly increased in FOXG1-derived neurons. To compare the relative amounts of excitatory vs. inhibitory synapses, the expression of related gene markers were analyzed; those associated with inhibitory synapses were increased, whereas those with excitatory synapses were decreased. The authors propose that this imbalance may be contributing to the neurodevelopmental features observed in FOXG1 syndrome.

Developed the ‘Developmental Encephalopathy Inventory’, a tool to measure and differentiate between different features of individuals with autism spectrum disorders or intellectual disabilities. The DEI is designed to evaluate fourteen different domains, including: ambulation, fine motor skills, language, mood, movements, seizures, sensory functions, etc. The authors used the DEI to compare features of individuals with FOXG1 syndrome and Rett syndrome. For both disorders, fine motor and expressive language skills were often impaired. The DEI was able to identify features that distinguished the two syndromes; generally, FOXG1 syndrome was assessed as being more severe, whereas those with Rett syndrome tended to become more severely affected with age.

The authors characterized video recordings of movement disorders in 8 children with FOXG1 syndrome. The observed movement disorder was described as ‘complex’, with combinations of dyskinetic and hyperkinetic movements, like
dystonia, chorea, and athetosis.

A mouse lacking Foxg1 expression was generated by replacing the coding sequencing with a lacZ/neomycin cassette; heterozygotes (only one copy of Foxg1 absent) were fertile and indistinguishable from wild-type littermates. Homozygotes were live born, but flaccid, with minimal spontaneous movements, and died shortly after birth.