FOXG1 targets BMP repressors and cell cycle inhibitors in human neural progenitor cells.

FOXG1 is a critical transcription factor in human brain where loss-of-function mutations cause a severe neurodevelopmental disorder, while increased FOXG1 expression is frequently observed in glioblastoma. FOXG1 is an inhibitor of cell patterning and an activator of cell proliferation in chordate model organisms but different mechanisms have been proposed as to how this occurs. To identify genomic targets of FOXG1 in human neural progenitor cells (NPCs), we engineered a cleavable reporter construct in endogenous FOXG1 and performed chromatin immunoprecipitation (ChIP) sequencing. We also performed deep RNA sequencing of NPCs from two females with loss-of-function mutations in FOXG1 and their healthy biological mothers. Integrative analyses of RNA and ChIP sequencing data showed that cell cycle regulation and Bone Morphogenic Protein (BMP) repression gene ontology categories were over-represented as FOXG1 targets. Using engineered brain cell lines, we show that FOXG1 specifically activates SMAD7 and represses CDKN1B. Activation of SMAD7 which inhibits BMP signaling may be one way that FOXG1 patterns the forebrain, while repression of cell cycle regulators such as CDKN1B may be one way that FOXG1 expands the NPC pool to ensure proper brain size. Our data reveal novel mechanisms on how FOXG1 may control forebrain patterning and cell proliferation in human brain development.

Kabuki syndrome stem cell models reveal locus specificity of histone methyltransferase 2D (KMT2D/MLL4).

Kabuki syndrome is frequently caused by loss-of-function mutations in one allele of histone 3 lysine 4 (H3K4) methyltransferase KMT2D and is associated with problems in neurological, immunological and skeletal system development. We generated heterozygous KMT2D knockout and Kabuki patient-derived cell models to investigate the role of reduced dosage of KMT2D in stem cells. We discovered chromosomal locus-specific alterations in gene expression, specifically a 110 Kb region containing Synaptotagmin 3 (SYT3), C-Type Lectin Domain Containing 11A (CLEC11A), Chromosome 19 Open Reading Frame 81 (C19ORF81) and SH3 And Multiple Ankyrin Repeat Domains 1 (SHANK1), suggesting locus-specific targeting of KMT2D. Using whole genome histone methylation mapping, we confirmed locus-specific changes in H3K4 methylation patterning coincident with regional decreases in gene expression in Kabuki cell models. Significantly reduced H3K4 peaks aligned with regions of stem cell maps of H3K27 and H3K4 methylation suggesting KMT2D haploinsufficiency impact bivalent enhancers in stem cells. Preparing the genome for subsequent differentiation cues may be of significant importance for Kabuki-related genes. This work provides a new insight into the mechanism of action of an important gene in bone and brain development and may increase our understanding of a specific function of a human disease-relevant H3K4 methyltransferase family member.

FOXG1 dose tunes cell proliferation dynamics in human forebrain progenitor cells.

Heterozygous loss-of-function mutations in Forkhead box G1 (FOXG1), a uniquely brain-expressed gene, cause microcephaly, seizures, and severe intellectual disability, whereas increased FOXG1 expression is frequently observed in glioblastoma. To investigate the role of FOXG1 in forebrain cell proliferation, we modeled FOXG1 syndrome using cells from three clinically diagnosed cases with two sex-matched healthy parents and one unrelated sex-matched control. Cells with heterozygous FOXG1 loss showed significant reduction in cell proliferation, increased ratio of cells in G0/G1 stage of the cell cycle, and increased frequency of primary cilia. Engineered loss of FOXG1 recapitulated this effect, while isogenic repair of a patient mutation reverted output markers to wild type. An engineered inducible FOXG1 cell line derived from a FOXG1 syndrome case demonstrated that FOXG1 dose-dependently affects all cell proliferation outputs measured. These findings provide strong support for the critical importance of FOXG1 levels in controlling human brain cell growth in health and disease.

Lesch-Nyhan disease causes impaired energy metabolism and reduced developmental potential in midbrain dopaminergic cells.

Mutations in HPRT1, a gene encoding a rate-limiting enzyme for purine salvage, cause Lesch-Nyhan disease which is characterized by self-injury and motor impairments. We leveraged stem cell and genetic engineering technologies to model the disease in isogenic and patient-derived forebrain and midbrain cell types. Dopaminergic progenitor cells deficient in HPRT showed decreased intensity of all developmental cell-fate markers measured. Metabolic analyses revealed significant loss of all purine derivatives, except hypoxanthine, and impaired glycolysis and oxidative phosphorylation. real-time glucose tracing demonstrated increased shunting to the pentose phosphate pathway for de novo purine synthesis at the expense of ATP production. Purine depletion in dopaminergic progenitor cells resulted in loss of RHEB, impairing mTORC1 activation. These data demonstrate dopaminergic-specific effects of purine salvage deficiency and unexpectedly reveal that dopaminergic progenitor cells are programmed to a high-energy state prior to higher energy demands of terminally differentiated cells.

Stimulation of L-type calcium channels increases tyrosine hydroxylase and dopamine in ventral midbrain cells induced from somatic cells.

Making high-quality dopamine (DA)-producing cells for basic biological or small molecule screening studies is critical for the development of novel therapeutics for disorders of the ventral midbrain. Currently, many ventral midbrain assays have low signal-to-noise ratio due to low levels of cellular DA and the rate-limiting enzyme of DA synthesis, tyrosine hydroxylase (TH), hampering discovery efforts. Using intensively characterized ventral midbrain cells derived from human skin, which demonstrate calcium pacemaking activity and classical electrophysiological properties, we show that an L-type calcium agonist can significantly increase TH protein levels and DA content and release. Live calcium imaging suggests that it is the immediate influx of calcium occurring simultaneously in all cells that drives this effect. Genome-wide expression profiling suggests that L-type calcium channel stimulation has a significant effect on specific genes related to DA synthesis and affects expression of L-type calcium receptor subunits from the CACNA1 and CACNA2D families. Together, our findings provide an advance in the ability to increase DA and TH levels to improve the accuracy of disease modeling and small molecule screening for disorders of the ventral midbrain, including Parkinson's disease.

FOXG1 Dose in Brain Development.

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.

Mutations in ACTL6B Cause Neurodevelopmental Deficits and Epilepsy and Lead to Loss of Dendrites in Human Neurons.

We identified individuals with variations in ACTL6B, a component of the chromatin remodeling machinery including the BAF complex. Ten individuals harbored bi-allelic mutations and presented with global developmental delay, epileptic encephalopathy, and spasticity, and ten individuals with de novo heterozygous mutations displayed intellectual disability, ambulation deficits, severe language impairment, hypotonia, Rett-like stereotypies, and minor facial dysmorphisms (wide mouth, diastema, bulbous nose). Nine of these ten unrelated individuals had the identical de novo c.1027G>A (p.Gly343Arg) mutation. Human-derived neurons were generated that recaptured ACTL6B expression patterns in development from progenitor cell to post-mitotic neuron, validating the use of this model. Engineered knock-out of ACTL6B in wild-type human neurons resulted in profound deficits in dendrite development, a result recapitulated in two individuals with different bi-allelic mutations, and reversed on clonal genetic repair or exogenous expression of ACTL6B. Whole-transcriptome analyses and whole-genomic profiling of the BAF complex in wild-type and bi-allelic mutant ACTL6B neural progenitor cells and neurons revealed increased genomic binding of the BAF complex in ACTL6B mutants, with corresponding transcriptional changes in several genes including TPPP and FSCN1, suggesting that altered regulation of some cytoskeletal genes contribute to altered dendrite development. Assessment of bi-alleic and heterozygous ACTL6B mutations on an ACTL6B knock-out human background demonstrated that bi-allelic mutations mimic engineered deletion deficits while heterozygous mutations do not, suggesting that the former are loss of function and the latter are gain of function. These results reveal a role for ACTL6B in neurodevelopment and implicate another component of chromatin remodeling machinery in brain disease.

BDNF Val66Met polymorphism and clinical response to antipsychotic treatment in schizophrenia and schizoaffective disorder patients: a meta-analysis.

Brain-derived neurotrophic factor (BDNF) plays an important role in dopaminergic and serotonergic neurotransmission by modulating dopaminergic neuron differentiation and establishment. Multiple studies have analyzed the functional BDNF Val66Met variant in relation to antipsychotic response in schizophrenia (SCZ) patients, yielding mixed results. A meta-analysis was thus performed to examine the relationship between this variant and symptom improvement during antipsychotic treatment. Searches using PubMed, Web of Science, and PsycInfo until October 2017 yielded 11 studies that met inclusion criteria (total n = 3774). These studies investigated the BDNF Val66Met variant and antipsychotic response in patients with SCZ or schizoaffective disorder. Responders to antipsychotics were defined using the original criteria applied in each study. Effect sizes were computed using odds ratios, which were pooled according to the Mantel-Haenszel method. The BDNF Val66Met variant was not associated with the total number of responders and non-responders (p > 0.05) under dominant, recessive, or allelic models. Secondary analyses stratifying for individuals of each ethnicity and drug type also revealed no significant associations. Our findings suggest that the BDNF Val66Met variant is not associated with response to antipsychotics in individuals with SCZ. However, considering the current sample size, small effects cannot be ruled out. Moreover, recent studies have suggested that Val66Met forms haplotypes with other BDNF variants. Future studies should examine the Val66Met variant in conjunction with these other variants in relation to antipsychotic response. Moreover, since illness duration appears to influence BDNF levels in SCZ patients, future studies should aim to control for this potential confounding factor in response analyses.

The effect of ethnicity and immigration on treatment resistance in schizophrenia.

BACKGROUND: Treatment resistance is a common issue among schizophrenia patients undergoing antipsychotic treatment. According to the American Psychiatric Association (APA) guidelines, treatment-resistant status is defined as little or no symptom reduction to at least two antipsychotics at a therapeutic dose for a trial of at least six weeks. The aim of the current study is to determine whether ethnicity and migration are associated with treatment resistance. METHODS: In a sample of 251 participants with schizophrenia spectrum disorders, we conducted cross-sectional assessments to collect information regarding self-identified ethnicity, immigration and treatment history. Ancestry was identified using 292 markers overlapping with the HapMap project. Using a regression analysis, we tested whether a history of migration, ethnicity or genetic ancestry were predictive of treatment resistance. RESULTS: Our logistic regression model revealed no significant association between immigration (OR = 0.04; 95%CI = 0.35-3.07; p = 0.93) and treatment resistant schizophrenia. White Europeans did not show significant association with resistance status regardless of whether ethnicity was determined by self-report (OR = 1.89; 95%CI = 0.89-4.20; p = 0.105) or genetic analysis (OR = -0.73; 95%CI = -0.18-2.97; p = 0.667). CONCLUSION: Neither ethnicity nor migrant status was significantly associated with treatment resistance in this Canadian study. However, these conclusions are limited by the small sample size of our investigation.

Differentiation of Human Induced Pluripotent Stem Cells (iPSCs) into an Effective Model of Forebrain Neural Progenitor Cells and Mature Neurons.

Induced Pluripotent Stem Cells (iPSCs) are pluripotent stem cells that can be generated from somatic cells, and provide a way to model the development of neural tissues in vitro. One particularly interesting application of iPSCs is the development of neurons analogous to those found in the human forebrain. Forebrain neurons play a central role in cognition and sensory processing, and deficits in forebrain neuronal activity contributes to a host of conditions, including epilepsy, Alzheimer's disease, and schizophrenia. Here, we present our protocol for differentiating iPSCs into forebrain neural progenitor cells (NPCs) and neurons, whereby neural rosettes are generated from stem cells without dissociation and NPCs purified from rosettes based on their adhesion, resulting in a more rapid generation of pure NPC cultures. Neural progenitor cells can be maintained as long-term cultures, or differentiated into forebrain neurons. This protocol provides a simplified and fast methodology of generating forebrain NPCs and neurons, and enables researchers to generate effective in vitro models to study forebrain disease and neurodevelopment. This protocol can also be easily adapted to generate other neural lineages.

A biopsychosocial evaluation of the risk for suicide in schizophrenia.

The risk of suicide is greatly increased in individuals with schizophrenia. Previous research has identified several potential risk factors for suicidal behavior in schizophrenia, although their ability to independently predict suicide is limited. The objective of this review was to systematically analyze and identify the interaction between the proposed risk factors in the literature that may predict suicidal behavior in schizophrenia. Articles that explored suicidal behavior and suicide risk in schizophrenia that were published between 1980 and August of 2015, indexed in PubMed, MEDLINE, and Scopus were systematically reviewed. Many studies proposed a range of biopsychosocial risk factors that may independently lead to suicide in schizophrenia. These risk factors appear to be mainly related to stress, a history of suicidal behavior, and psychotic symptoms. It is clear, however, that many of these factors do not act independently and in fact require the reciprocal interaction of several of them to pose a risk for suicide in schizophrenia. Independently, the power of many risk factors to predict suicide is limited. Future studies should continue to adopt a multidimensional approach by considering the interaction of several factors in assessing the risk for suicide in schizophrenia.

Strategies to Advance Drug Discovery in Rare Monogenic Intellectual Disability Syndromes.

Some intellectual disability syndromes are caused by a mutation in a single gene and have been the focus of therapeutic intervention attempts, such as Fragile X and Rett Syndrome, albeit with limited success. The rate at which new drugs are discovered and tested in humans for intellectual disability is progressing at a relatively slow pace. This is particularly true for rare diseases where so few patients make high-quality clinical trials challenging. We discuss how new advances in human stem cell reprogramming and gene editing can facilitate preclinical study design and we propose new workflows for how the preclinical to clinical trajectory might proceed given the small number of subjects available in rare monogenic intellectual disability syndromes.

Validating a research ethnicity questionnaire using genomic markers.

AIM: Population stratification is a confounding factor in genetic association studies. Self-report measures, the most common method of collecting ethnicity, may be less reliable for psychiatric patients. This study aims to validate our research ethnicity questionnaire as a reliable measure of genetic ancestry. METHODS: The validity of our questionnaire was compared with genetic ancestry according to structured association tests and dimensional reduction methods. Our research tool was also compared with a standard multiple choice questionnaire. RESULTS: Our research questionnaire was highly consistent with genetic ancestry. The standard questionnaire demonstrated a greater degree of inconsistency in identifying ethnicity. CONCLUSION: Collecting information on the geographical ancestry of each individual's grandparents provides a more comprehensive view of ethnicity to prevent population stratification and wasted finances on genotyping.

Classification of suicide attempters in schizophrenia using sociocultural and clinical features: A machine learning approach.

OBJECTIVE: Suicide is a major concern for those afflicted by schizophrenia. Identifying patients at the highest risk for future suicide attempts remains a complex problem for psychiatric interventions. Machine learning models allow for the integration of many risk factors in order to build an algorithm that predicts which patients are likely to attempt suicide. Currently it is unclear how to integrate previously identified risk factors into a clinically relevant predictive tool to estimate the probability of a patient with schizophrenia for attempting suicide. METHODS: We conducted a cross-sectional assessment on a sample of 345 participants diagnosed with schizophrenia spectrum disorders. Suicide attempters and non-attempters were clearly identified using the Columbia Suicide Severity Rating Scale (C-SSRS) and the Beck Suicide Ideation Scale (BSS). We developed four classification algorithms using a regularized regression, random forest, elastic net and support vector machine models with sociocultural and clinical variables as features to train the models. RESULTS: All classification models performed similarly in identifying suicide attempters and non-attempters. Our regularized logistic regression model demonstrated an accuracy of 67% and an area under the curve (AUC) of 0.71, while the random forest model demonstrated 66% accuracy and an AUC of 0.67. Support vector classifier (SVC) model demonstrated an accuracy of 67% and an AUC of 0.70, and the elastic net model demonstrated and accuracy of 65% and an AUC of 0.71. CONCLUSION: Machine learning algorithms offer a relatively successful method for incorporating many clinical features to predict individuals at risk for future suicide attempts. Increased performance of these models using clinically relevant variables offers the potential to facilitate early treatment and intervention to prevent future suicide attempts.

GWAS analysis of treatment resistant schizophrenia: interaction effect of childhood trauma.

AIMS: In the current study, we aimed to compare the prevalence of adverse lifetime events in treatment resistant and non-treatment resistant schizophrenia in a genome-wide association study. MATERIALS & METHODS: Our sample consisted of 84 Caucasian participants with schizophrenia spectrum disorders, assessed cross-sectionally to collect information regarding drug effectiveness and childhood trauma. Using a genome-wide association analysis, we tested single-nucleotide polymorphisms for their association with resistance to antipsychotics defined according to American Psychiatric Association criteria. Two models were tested: a main model and an interaction model with the childhood trauma. RESULTS: Our analysis failed to demonstrate a significant relationship among 1,178,234 single-nucleotide polymorphisms and treatment-resistance in both the main model and in the childhood trauma interaction model. CONCLUSION: Even though we could not find any significant association, treatment resistance has clinical relevance and it may be determined by the interaction between biological and non biological factors.

Assessing the risk for suicide in schizophrenia according to migration, ethnicity and geographical ancestry.

BACKGROUND: Suicide is a leading cause of mortality among those afflicted by schizophrenia. Previous studies demonstrated that the stressors associated with immigration may lead to an onset of schizophrenia and suicide separately in susceptible individuals. However, no studies have shown whether immigration may lead to suicidal behaviour for individuals with schizophrenia. Our study proposes that an individual's geographical ancestry, ethnicity or migration status may be predictive of suicide risk in schizophrenia. METHODS: In a sample of 276 participants with schizophrenia spectrum disorders, we conducted cross-sectional assessments to collect clinical information. Self-identified ethnicity and suicide history were collected through self-report questionnaires and interview-based scales. Ancestry was identified using 292 genetic markers from HapMap. Migrants were classified as those who immigrated to Canada during their lifetime. Using a regression analysis, we tested whether a history of migration, ethnicity or geographical ancestry were predictive of a history of suicide attempts. RESULTS: Our analysis failed to demonstrate a significant relationship between suicide history and migration, ethnicity or ancestry. However, ethnicity appears to be significantly associated with the number of psychiatric hospitalizations in our sample. CONCLUSION: Ethnicity and migration history are not predictive of previous suicide attempts. Ethnicity may be an important demographic factor affecting access to mental health resources and frequency of hospitalizations.

Candidate gene analysis of pharmacodynamic targets for antipsychotic dosage.

AIM: In order to administer antipsychotic medication with the most beneficial outcome, the appropriate drug and dose needs to be identified. Though often not considered in pharmacogenetic studies, dosage plays an important role in treatment outcome. This study set out to analyze the association between 109 SNPs and antipsychotic dosage among schizophrenia patients. In a previous study, we tested 134 SNPs in regards to antipsychotic dosage. In the current study, we tested additional markers in the same candidate genes that we investigated in the previous study to confirm our previous findings. METHODS: We included 263 participants with schizophrenia spectrum disorders between the ages of 18-75. Each participant was assessed cross-sectionally to collect clinical and antipsychotic treatment information through a semi-structured interview. The antipsychotic dosage for each individual was standardized according to chlorpromazine equivalents (CPZe), defined daily dose, and the percentage of maximum dosage (PM%). For each participant, 109 SNPs from 29 candidate genes were imputed or genotyped using a Customized Illumina Chip. RESULTS: Polymorphisms in the GABRB1 gene were significantly associated with higher antipsychotic dosage according to CPZe and PM% standardization. CONCLUSION: Our analysis suggests that variation in the GABRB1 gene may be significantly associated with antipsychotic dosage according to CPZe and PM% standardization. Antipsychotic dosage remains an integral measure for treatment response that warrants future pharmacogenetic testing and studies with larger sample sizes.

Polygenic risk score prediction of antipsychotic dosage in schizophrenia.

OBJECTIVE: Genetic variants have yet to be identified as reliable predictors of antipsychotic dosage. The purpose of this study is to quantify significant genetic risk variants prioritized from the Psychiatric GWAS Consortium (PGC2) study for schizophrenia as a polygenic score to test our hypothesis that it may represent symptom severity in patients and therefore predict antipsychotic dosage. METHODS: Antipsychotic medication and dosage were collected in our sample of 83 patients with schizophrenia spectrum disorders of a homogeneous European background. Antipsychotic dosage was standardized according to the Product Monograph (PM%), chlorpromazine equivalents (CPZe), and Defined Daily Dose (DDD). We calculated polygenic risk scores (PRS) for the significant risk loci identified from the PGC2 GWAS to predict dosage using a linear regression model. RESULTS: In our analysis, the PRS showed no significant association with PM%, CPZe, and DDD dosage. Considering symptom severity and overall functioning, our PRS was similarly not significantly associated with Global Assessment of Functioning (GAF) scores. DISCUSSION: Our results do not provide evidence for a polygenic inheritance of schizophrenia that influences levels of antipsychotic dosage. To the best of our knowledge, this is one of the first studies of its kind to use the PRS from the PGC2 significant risk variants to predict a clinically relevant phenotype. The PRS offers a novel approach to analyzing the genetic liability for many clinically relevant phenotypes in schizophrenia.

Use of candidate gene markers to guide antipsychotic dosage adjustment.

OBJECTIVE: To improve antipsychotic treatment in schizophrenia patients, many studies have investigated genetic polymorphisms associated with antipsychotic metabolizing enzymes and receptors. While these studies have typically focused on drug response, few have investigated genetic influences on antipsychotic dosage. This study set out to analyze the association between 134 SNPs in 38 candidate genes and antipsychotic dosage in schizophrenia patients. METHODS: For our analysis, 300 patients with a diagnosis of either schizophrenia or schizoaffective disorder were recruited between the ages of 18 and 75. A cross-sectional assessment was used, in which data were collected from each participant through an interview and self-report questionnaire. Antipsychotic dose was standardized according to the chlorpromazine equivalents, defined daily dose and relative to the maximum dose specified in the product monograph. Participants were genotyped using a Customized Illumina Chip comprising 134 SNPs, and all markers were screened for nominal significance. RESULTS: The analysis showed a nominally significant association with the GFRA1 gene. CONCLUSION: The common variants investigated in this study had no major influence on the antipsychotic dosage prescribed in study participants. It remains, though, that this strategy may prove valuable clinically and warrants further investigation.

Does a history of suicide attempt predict higher antipsychotic dosage in schizophrenia?

RATIONALE: Antipsychotic dosage is generally adjusted by physicians depending on the stability of the patient and the response to that particular drug. Our hypothesis is that patients with previous suicide attempt are prescribed higher doses of antipsychotics. OBJECTIVE: We examined the dosage and patterns of antipsychotic use in regard to past suicidal behaviour. METHODS: For this study, 304 subjects with schizophrenia spectrum disorders between the ages of 18 and 75 were recruited. A cross-sectional assessment was used for this study, in which data were collected from each patient through an interview and self-report questionnaires. The percentages of the Compendium of Pharmaceuticals and Specialties (CPS) maximum recommended daily dose were applied to standardize antipsychotic dosages across different treatments. We compared the standardized dosage of antipsychotics in schizophrenics with previous suicide attempts and those who have never attempted suicide. RESULTS: Applying the ANCOVA, our preliminary results show no significant difference (P = 0.467) in antipsychotic dosage in the attempters and non-attempters. The prescribed clozapine dosage fails to show a significant relationship with suicidal history (P >0.05). CONCLUSIONS: In summary, our analysis does not show antipsychotic dosage adjustment based on past suicide attempt, after controlling for the current suicidal ideation and hopelessness.