Expanding genotype-phenotype correlations in FOXG1 syndrome: results from a patient registry

Authors: Elise Brimble, Kathryn G Reyes, Kopika Kuhathaas, Orrin Devinsky, Maura R Z Ruzhnikov, Xilma R Ortiz-Gonzalez, Ingrid Scheffer, Nadia Bahi-Buisson, Heather Olson; FOXG1 Research Foundation

Journal: Orphanet Journal of Rare Diseases

Date: June 12, 2023

Abstract:

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.

Results: We studied 122 registry participants with FOXG1 syndrome, aged < 12 months to 24 years. Caregivers described delayed or absent developmental milestone attainment, seizures (61%), and movement disorders (58%). Participants harbouring a missense variant had a milder phenotype. Compared to individuals with gene deletions (0%) or nonsense variants (20%), missense variants were associated with more frequent attainment of sitting (73%). Further, individuals with missense variants (41%) achieved independent walking more frequently than those with gene deletions (0%) or frameshift variants (6%). Presence of epilepsy also varied by genotype and was significantly more common in those with gene deletions (81%) compared to missense variants (47%). Individuals with gene deletions were more likely to have higher seizure burden than other genotypes with 53% reporting daily seizures, even at best control. We also observed that truncations preserving the forkhead DNA binding domain were associated with better developmental outcomes.

Conclusion: We refine the phenotypic spectrum of neurodevelopmental features associated with FOXG1 syndrome. We strengthen genotype-driven outcomes, where missense variants are associated with a milder clinical course.

The clinical and sleep manifestations in children with FOXG1 syndrome

Authors: Lee Chin-Wong, Cheng-Hsien Huang, Wan-Yun Chou, Chia-Jui Hsu, Wen-Che Tsai, Wang-Tso Lee

Journal: Autism Research INSAR

Date: March 21, 2023

Abstract:

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.

We analyzed the clinical and sleep manifestations of children with FOXG1 syndrome registered in the FOXG1 Research Foundation Patient Registry database. A total of 258 individuals with FOXG1 syndrome were included in this research. 132 (51.16%) had sleep disturbances. The more impaired of language acquisitions (absence of speech, OR: 3.99, 95%CI = 1.69–9.42, p = 0.002), hyperkinetic movement disorders (OR: 2.64, 95%CI = 1.34-5.20 p = 0.005) and feeding difficulties (OR: 2.81, 95% CI = 1.52–5.19, p = 0.001) were significantly associated with an increase in odds of sleep disturbance after adjusting for age, sex, and antiepileptic drugs.

We also performed sleep studies on six individuals with FOXG1 syndrome using The Children's Sleep Habits Questionnaire (CSHQ), the Sleep Disturbance Scale for Children (SDSC), and 7-day data from Actiwatch. The Pittsburgh Sleep Quality Index (PSQI) and 7-day data from Actiwatch were also used to evaluate the sleep condition of their parents. The CSHQ scores revealed bedtime resistance, sleep onset delay, sleep duration, sleep anxiety, night-waking, and parasomnia. Sleep–wake transition disorders and disorders of initiating and maintaining sleep were also suggested by the SDSC scores. The children's actigraphy revealed short sleep durations, impaired sleep efficiency, longer wake after sleep onset, and frequent night-waking.

All caregivers reported significantly higher PSQI scores, mildly declined sleep efficiency, and shorter total sleep duration. Sleep disturbances, especially in initiating and maintaining sleep, are common in individuals with FOXG1 syndrome and their caregivers. Sleep disorders in patients with FOXG1 syndrome and their caregivers should be investigated.

Behavioral Phenotypes of Foxg1 Heterozygous Mice

Authors: Skyler Younger, Sydney Boutros, Francesca Cargnin, Shin Jeon,, Jae W. Lee, Soo-Kyung Lee and Jacob Raber

Journal: Frontiers in Pharmacology, section Neuropharmacology.

Date: June 8, 2022

Abstract:

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.

Significance:

This paper from Lee lab at University at Buffalo, conducted in collaboration with Jacob Raber’s group at Oregon Health & Science University, is the first comprehensive behavioral studies on Foxg1 heterozygous mice.  They found that Foxg1 heterozygous mice show various phenotypes resembling the symptoms of the human FOXG1 syndrome individuals.  Those 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.  The results presented in this paper suggest that Foxg1 heterozygous mice recapitulate at least some symptoms of the human FOXG1 syndrome individuals.

However, it is important to note that the spectrum of symptoms widely differs among FOXG1 syndrome individuals depending on the type and location of the mutation in the FOXG1 gene, highlighting the need for patient-specific animal models and personalized therapeutic intervention for FOXG1 syndrome individuals.  Lee lab has also been developing a series of patient-specific mouse models.  Excitingly, their preliminary results reveal that those mouse models faithfully recapitulate various patient-specific symptoms. These new mouse models will serve as the fundamental platforms to study the etiology of the FOXG1 syndrome and for drug discovery.

Funding:

This work was partly supported by the FOXG1 Research Foundation, S-KL, and JL.

FoxG1 regulates the formation of cortical GABAergic circuit during an early postnatal critical period resulting in autism spectrum disorder-like phenotypes

First Author: Goichi Miyoshi (More authors)

Journal: Nature Communications

Year: 2021

Abstract

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.

Significance:

New understanding of FOXG1: Increased or decreased FoxG1 expression in both excitatory and inhibitory neurons results in ASD-related circuit and social behavior deficits in mouse models and and may participate in regulation of mitochondrial function. Goichi identifies both the critical time window and the inhibitory circuit mechanisms that lead to ASD-related behavior.

Precisely controlling endogenous protein dosage in hPSCs and derivatives to model FOXG1 syndrome

First Author: Wenliang Zhu (More authors)
Journal: Nature Communications
Year: 2019

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.

Significance
A new study from State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, and Chinese Academy of Sciences, Beijing, China shows how SMASh enables tunable shut-off of transgene in hPSCs

Discovery of the genetic 'conductor' of brain stem cells

First Author: Carmen Falcone (More authors)
Journal: Celebral Cortex
Year: 2019

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.

Significance
A new study by SISSA identifies the gene which regulates the production ratio of neurons and support cells during cerebral cortex development..

FOXG1 Dose in Brain Development

Authors: Nuwan C. Hettige, McGill University 
Journal: Frontiers in Pediatrics
Year: 2019

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.

FOXG1 Orchestrates Neocortical Organization and Cortico-Cortical Connections

First Author: Francesca Cargnin (More authors) including Soo-Kyung Lee
Journal: Neuron
Year: 2018

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.

Significance
Study reveals a novel gene regulatory pathway that specifies neuronal characteristics during cerebral cortex development and sheds light on the etiology of FOXG1 syndrome.

Delineating FOXG1 syndrome From congenital microcephaly to hyperkinetic encephalopathy

First Author: Nancy Vegas, MD (More authors)
Journal: Neurology Genetics
Year: 2018

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 first symptoms most commonly reported in the 45 new cases included: developmental delay, microcephaly (small head size), and differences in visual tracking. By childhood, FOXG1 syndrome is characterized by: developmental and intellectual disabilities, limited functional hand use, and a complex movement disorder. Seizures are reported in ~78%, with age of onset ranging from shortly after birth to childhood; in about half, seizures are not controlled by medication. An important part of this case series focused on brain imaging findings in people with FOXG1 syndrome. The authors describe three main abnormalities: (1) delayed myelination, (2) abnormal gyri, and (3) abnormal corpus callosum. Myelination, which continues even after birth, is an important process that helps neurons transmit information. Brain imaging in FOXG1 syndrome shows that this process may occur more slowly. The gyri are the folds of the brain; in some people with FOXG1 syndrome, there can be fewer folds in the front part of the brain. The corpus callosum is a large tract that connects the two hemispheres of the brain. In FOXG1 syndrome, this tract can look smaller or may be absent. Importantly, in a small number of cases who had brain imaging earlier and later in life, there is interval development. Finally, information from this study supports other work that suggests mutations towards the beginning of the FOXG1 gene, or complete absence of the FOXG1 gene, may be associated with more severe symptoms.

Significance As more cases are published in the medical literature, our understanding of FOXG1 syndrome grows. Knowing the number of people with FOXG1 syndrome and identifying clinical endpoints are important tools for developing treatments.

Phenotype differentiation of FOXG1 and MECP2 disorders: a new method for characterization of developmental encephalopathies

First Author: Mandy Ma (More authors)
Journal: The Journal of Pediatrics
Year: 2016

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.

Significance
Developed a systematic way to evaluate clinical severity in individuals with FOXG1 syndrome. This can be used to measure the success or limitations of proposed therapies in clinical trials.

The hyperkinetic movement disorder of FOXG1-related epileptic-dyskinetic encephalopathy

First Author: Elena Cellini (More authors)
Journal: Developmental Medicine & Child Neurology
Year: 2016

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.

Significance
Provides improved characterization of the movement disorders in individuals with FOXG1 syndrome, which may be important for medical management.

FOXG1 syndrome: genotype-phenotype association in 83 patients with FOXG1 variants

First Author: Diana Mitter (More authors)
Journal: Genetics in Medicine
Year: 2016

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).

A range of epilepsy diagnoses were reported, with a wide range in the age of onset (3 to 168 months). There does not seem to be a characteristic seizure type.

Importantly, 5 families have now been documented with presumed parental mosaicism; the authors recommend that prenatal genetic diagnosis be offered to families with a child who has FOXG1 syndrome.

Significance
First publication demonstrating genotype-phenotype correlations (i.e. the type of FOXG1 mutation impacts clinical severity), and uses a new standardized assessment of clinical severity in the largest published population of individuals with FOXG1 syndrome.

The role of FOXG1 in brain development and function

FOXG1-dependent dysregulation of GABA/Glutamatergic neuron differentiation in autism spectrum disorders

First Author: Jessica Mariani (More authors)
Journal: Cell
Year: 2015

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.

In ASD, larger head size has been associated with more severe symptoms and lower IQ; in 9 individuals with ASD and a larger head size; increased FOXG1 expression correlated with greater head size.

Significance
Implicates FOXG1 dysregulation in the pathogenesis of autism spectrum disorders, and possible in head size.

Imbalance of excitatory/inhibitory synaptic protein expression in iPSC-derived neurons from FOXG1 patients and in foxg1 mice

First Author: Tommaso Patriarchi (More authors)
Journal: European Journal of Human Genetics
Year: 2016

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

Significance
First published use of iPSC neurons derived from individuals with FOXG1 syndrome; a phenotype is identified, which can be used as a marker in future experiments to measure the success of potential therapies.

Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres

First Author: Shouhong Xuan (More authors)
Journal: Neuron
Year: 1995

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.

Homozygotes were noted to have a significant reduction in brain size. The ventral telencephalon was noted to be more severely affected than the dorsal telencephalon; almost all telencephalon examined at E12.5 expressed dorsal markers, and not ventral markers. The authors further demonstrate that at E10.5, only dorsal telencephalic neuroepithelial cells are actively proliferating – ventral telencephalon cells are not. Authors suggest that their data demonstrate that absence of Foxg1 results in early withdrawal of telencephalic NPCs from the cell cycle, and premature onset of neuronal differentiation.

Significance
First Foxg1 animal model published; associated differences in brain development well-documented in the homozygous state.

RNA activation of haploinsufficient Foxg1 gene in murine neocortex

First Author: Cristina Fimiani (More authors)
Journal: Scientific Reports
Year: 2016

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.

Delivery of a similar miR molecule using an AAV9 system into newly born mouse pups showed modest upregulation of Foxg1 expression in the brain.

Significance
Demonstrates efficacy of using RNA activation to boost endogenous expression of Foxg1; this is a potential therapeutic strategy to alleviate clinical symptoms of FOXG1 syndrome