Underrepresentation of Phenotypic Variability of 16p13.11 Microduplication Syndrome Assessed With an Online Self-Phenotyping Tool (Phenotypr): Cohort Study.

BACKGROUND: 16p13.11 microduplication syndrome has a variable presentation and is characterized primarily by neurodevelopmental and physical phenotypes resulting from copy number variation at chromosome 16p13.11. Given its variability, there may be features that have not yet been reported. The goal of this study was to use a patient "self-phenotyping" survey to collect data directly from patients to further characterize the phenotypes of 16p13.11 microduplication syndrome. OBJECTIVE: This study aimed to (1) discover self-identified phenotypes in 16p13.11 microduplication syndrome that have been underrepresented in the scientific literature and (2) demonstrate that self-phenotyping tools are valuable sources of data for the medical and scientific communities. METHODS: As part of a large study to compare and evaluate patient self-phenotyping surveys, an online survey tool, Phenotypr, was developed for patients with rare disorders to self-report phenotypes. Participants with 16p13.11 microduplication syndrome were recruited through the Boston Children's Hospital 16p13.11 Registry. Either the caregiver, parent, or legal guardian of an affected child or the affected person (if aged 18 years or above) completed the survey. Results were securely transferred to a Research Electronic Data Capture database and aggregated for analysis. RESULTS: A total of 19 participants enrolled in the study. Notably, among the 19 participants, aggression and anxiety were mentioned by 3 (16%) and 4 (21%) participants, respectively, which is an increase over the numbers in previously published literature. Additionally, among the 19 participants, 3 (16%) had asthma and 2 (11%) had other immunological disorders, both of which have not been previously described in the syndrome. CONCLUSIONS: Several phenotypes might be underrepresented in the previous 16p13.11 microduplication literature, and new possible phenotypes have been identified. Whenever possible, patients should continue to be referenced as a source of complete phenotyping data on their condition. Self-phenotyping may lead to a better understanding of the prevalence of phenotypes in genetic disorders and may identify previously unreported phenotypes.

16p13.11 deletion variants associated with neuropsychiatric disorders cause morphological and synaptic changes in induced pluripotent stem cell-derived neurons.

16p13.11 copy number variants (CNVs) have been associated with autism, schizophrenia, psychosis, intellectual disability, and epilepsy. The majority of 16p13.11 deletions or duplications occur within three well-defined intervals, and despite growing knowledge of the functions of individual genes within these intervals, the molecular mechanisms that underlie commonly observed clinical phenotypes remain largely unknown. Patient-derived, induced pluripotent stem cells (iPSCs) provide a platform for investigating the morphological, electrophysiological, and gene-expression changes that result from 16p13.11 CNVs in human-derived neurons. Patient derived iPSCs with varying sizes of 16p13.11 deletions and familial controls were differentiated into cortical neurons for phenotypic analysis. High-content imaging and morphological analysis of patient-derived neurons demonstrated an increase in neurite branching in patients compared with controls. Whole-transcriptome sequencing revealed expression level changes in neuron development and synaptic-related gene families, suggesting a defect in synapse formation. Subsequent quantification of synapse number demonstrated increased numbers of synapses on neurons derived from early-onset patients compared to controls. The identification of common phenotypes among neurons derived from patients with overlapping 16p13.11 deletions will further assist in ascertaining common pathways and targets that could be utilized for screening drug candidates. These studies can help to improve future treatment options and clinical outcomes for 16p13.11 deletion patients.

Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and their differentiation into neural lineages is a revolutionary experimental system for studying neurological disorders, including intellectual and developmental disabilities (IDDs). However, issues related to variability and reproducibility have hindered translating preclinical findings into drug discovery. Here, we identify areas for improvement by conducting a comprehensive review of 58 research articles that utilized iPSC-derived neural cells to investigate genetically defined IDDs. Based upon these findings, we propose recommendations for best practices that can be adopted by research scientists as well as journal editors.

Development of electrophysiological and morphological properties of human embryonic stem cell-derived GABAergic interneurons at different times after transplantation into the mouse hippocampus.

Transplantation of human embryonic stem cell (hESC)-derived neural progenitors is a potential treatment for neurological disorders, but relatively little is known about the time course for human neuron maturation after transplantation and the emergence of morphological and electrophysiological properties. To address this gap, we transplanted hESC-derived human GABAergic interneuron progenitors into the mouse hippocampus, and then characterized their electrophysiological properties and dendritic arborizations after transplantation by means of ex vivo whole-cell patch clamp recording, followed by biocytin staining, confocal imaging and neuron reconstruction software. We asked whether particular electrophysiological and morphological properties showed maturation-dependent changes after transplantation. We also investigated whether the emergence of particular electrophysiological properties were linked to increased complexity of the dendritic arbors. Human neurons were classified into five distinct neuronal types (Type I-V), ranging from immature to mature fast-spiking interneurons. Hierarchical clustering of the dendritic morphology and Sholl analyses suggested four morphologically distinct classes (Class A-D), ranging from simple/immature to highly complex. Incorporating all of our data regardless of neuronal classification, we investigated whether any electrophysiological and morphological features correlated with time post-transplantation. This analysis demonstrated that both dendritic arbors and electrophysiological properties matured after transplantation.

Examining the role of the surfactant family member SFTA3 in interneuron specification.

The transcription factor NKX2.1, expressed at high levels in the medial ganglionic eminence (MGE), is a master regulator of cortical interneuron progenitor development. To identify gene candidates with expression profiles similar to NKX2.1, previous transcriptome analysis of human embryonic stem cell (hESC)-derived MGE-like progenitors revealed SFTA3 as the strongest candidate. Quantitative real-time PCR analysis of hESC-derived NKX2.1-positive progenitors and transcriptome data available from the Allen Institute for Brain Science revealed comparable expression patterns for NKX2.1 and SFTA3 during interneuron differentiation in vitro and demonstrated high SFTA3 expression in the human MGE. Although SFTA3 has been well studied in the lung, the possible role of this surfactant protein in the MGE during embryonic development remains unexamined. To determine if SFTA3 plays a role in MGE specification, SFTA3-/- and NKX2.1 -/- hESC lines were generated using custom designed CRISPRs. We show that NKX2.1 KOs have a significantly diminished capacity to differentiate into MGE interneuron subtypes. SFTA3 KOs also demonstrated a somewhat reduced ability to differentiate down the MGE-like lineage, although not as severe relative to NKX2.1 deficiency. These results suggest NKX2.1 and SFTA3 are co-regulated genes, and that deletion of SFTA3 does not lead to a major change in the specification of MGE derivatives.

Pluripotent stem cell-derived interneuron progenitors mature and restore memory deficits but do not suppress seizures in the epileptic mouse brain.

GABAergic interneuron dysfunction has been implicated in temporal lobe epilepsy (TLE), autism, and schizophrenia. Inhibitory interneuron progenitors transplanted into the hippocampus of rodents with TLE provide varying degrees of seizure suppression. We investigated whether human embryonic stem cell (hESC)-derived interneuron progenitors (hESNPs) could differentiate, correct hippocampal-dependent spatial memory deficits, and suppress seizures in a pilocarpine-induced TLE mouse model. We found that transplanted ventralized hESNPs differentiated into mature GABAergic interneurons and became electrophysiologically active with mature firing patterns. Some mice developed hESNP-derived tumor-like NSC clusters. Mice with transplants showed significant improvement in the Morris water maze test, but transplants did not suppress seizures. The limited effects of the human GABAergic interneuron progenitor grafts may be due to cell type heterogeneity within the transplants.

Hedgehog Promotes Production of Inhibitory Interneurons in Vivo and in Vitro from Pluripotent Stem Cells.

Loss or damage of cortical inhibitory interneurons characterizes a number of neurological disorders. There is therefore a great deal of interest in learning how to generate these neurons from a pluripotent stem cell source so they can be used for cell replacement therapies or for in vitro drug testing. To design a directed differentiation protocol, a number of groups have used the information gained in the last 15 years detailing the conditions that promote interneuron progenitor differentiation in the ventral telencephalon during embryogenesis. The use of Hedgehog peptides and agonists is featured prominently in these approaches. We review here the data documenting a role for Hedgehog in specifying interneurons in both the embryonic brain during development and in vitro during the directed differentiation of pluripotent stem cells.

Transcriptome and in Vitro Differentiation Profile of Human Embryonic Stem Cell Derived NKX2.1-Positive Neural Progenitors.

The generation of inhibitory interneuron progenitors from human embryonic stem cells (ESCs) is of great interest due to their potential use in transplantation therapies designed to treat central nervous system disorders. The medial ganglionic eminence (MGE) is a transient embryonic structure in the ventral telencephalon that is a major source of cortical GABAergic inhibitory interneuron progenitors. These progenitors migrate tangentially to sites in the cortex and differentiate into a variety of interneuron subtypes, forming local synaptic connections with excitatory projection neurons to modulate activity of the cortical circuitry. The homeobox domain-containing transcription factor NKX2.1 is highly expressed in the MGE and pre-optic area of the ventral subpallium and is essential for specifying cortical interneuron fate. Using a combination of growth factor agonists and antagonists to specify ventral telencephalic fates, we previously optimized a protocol for the efficient generation of NKX2.1-positive MGE-like neural progenitors from human ESCs. To establish their identity, we now characterize the transcriptome of these MGE-like neural progenitors using RNA sequencing and demonstrate the capacity of these cells to differentiate into inhibitory interneurons in vitro using a neuron-astrocyte co-culture system. These data provide information on the potential origin of interneurons in the human brain.