Ellen J. Hoffman

Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

Anxiety Disorders: A Comprehensive Review of Pharmacotherapies

This article reviews the evidence from randomized, placebo-controlled trials and meta-analyses of pharmacological treatments of the following anxiety disorders: generalized anxiety disorder, panic disorder, social anxiety disorder, and post-traumatic stress disorder. There is evidence from multiple randomized, placebo-controlled trials to support the use of selective serotonin reuptake inhibitors as first-line pharmacotherapy in these disorders, and a number of the selective serotonin reuptake inhibitors have received US Food and Drug Administration approval for these indications. Serotonin-norepinephrine reuptake inhibitors are now emerging as first-line treatments for these anxiety disorders alongside the selective serotonin reuptake inhibitors and have been US Food and Drug Administration-approved for some of these indications as well. Benzodiazepines are also effective treatments for anxiety disorders, and although this medication class has the advantage of a rapid onset of action, their use is limited by their potential for abuse and lack of antidepressant properties. In addition to reviewing the clinical trials that have investigated the anxiolytic effects of these commonly used medications, we review the evidence for novel uses of other agents, including anticonvulsants and atypical antipsychotics, in anxiety disorders.

Multiplex ligation-dependent probe amplification for genetic screening in autism spectrum disorders: Efficient identification of known microduplications and identification of a novel microduplication in ASMT

BackgroundIt has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications.MethodsIn the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region.ResultsMLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, de novo duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include GABRB3 and ATP10A in one case, and MKRN3, MAGEL2 and NDN in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the ASPA gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the TM4SF2 gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the ASMT gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6–7% of the cases but in only 2% of controls (P = 0.003).ConclusionMLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new de novo small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in TM4SF2 are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the ASMT locus indicate that further studies of the duplication of the ASMT gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods.

Estrogens Suppress a Behavioral Phenotype in Zebrafish Mutants of the Autism Risk Gene, CNTNAP2

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

A large-scale screen for coding variants in SERT/SLC6A4 in autism spectrum disorders.

In the current study we explored the hypothesis that rare variants in SLC6A4 contribute to autism susceptibility and to rigid‐compulsive behaviors in autism. We made use of a large number of unrelated cases with autism spectrum disorders (∼350) and controls (∼420) and screened for rare exonic variants in SLC6A4 by a high‐throughput method followed by sequencing. We observed no difference in the frequency of such variants in the two groups, irrespective of how we defined the rare variants. Furthermore, we did not observe an association of rare coding variants in SLC6A4 with rigid‐compulsive traits scores in the cases. These results do not support a significant role for rare coding variants in SLC6A4 in autism spectrum disorders, nor do they support a significant role for SLC6A4 in rigid‐compulsive traits in these disorders.

Effects of Ethanol on Axon Outgrowth and Branching in Developing Rat Cortical Neurons

Humans exposed prenatally to ethanol can exhibit brain abnormalities and cognitive impairment similar to those seen in patients expressing mutant forms of the L1 cell adhesion molecule (L1CAM). The resemblance suggests that L1CAM may be a target for ethanol, and consistent with this idea, ethanol can inhibit L1CAM adhesion in cell lines and L1CAM-mediated outgrowth and signaling in cerebellar granule neurons. However, it is not known whether ethanol inhibits L1CAM function in other neuron types known to require L1CAM for appropriate development. Here we asked whether ethanol alters L1CAM function in neurons of the rat cerebral cortex. We find that ethanol does not alter axonal polarization, L1CAM-dependent axon outgrowth or branching, or L1CAM recycling in axonal growth cones. Thus, ethanol inhibition of L1CAM is highly dependent on neuronal context.

Progress in Cytogenetics: Implications for Child Psychopathology

OBJECTIVE This review considers the impact of chromosomal studies on the understanding of childhood neuropsychiatric syndromes, highlighting key discoveries, advances in technology, and new challenges faced by clinicians trying to interpret recent findings. METHOD We review the literature on the genetics of child psychiatric disorders, including autism, childhood-onset schizophrenia, attention-deficit/hyperactivity disorder, and Tourette syndrome, with a focus on studies of chromosomal structure. RESULTS Over several decades, cytogenetic investigations have led to key findings relevant to child psychiatry. During this time, technology has transitioned from light microscopy to molecular cytogenetics to microarray-based detection of structural variation, resulting in a dramatic increase in the resolution of such approaches. Each of these methods has contributed to the understanding of the genetic bases of developmental neuropsychiatric disorders. Moreover, the implementation of microarray technology has prompted a reconceptualization of the nature of human genetic variation, demonstrating that both the sequence of DNA as well as the fine structure of chromosomes vary in affected and unaffected individuals. CONCLUSIONS The study of chromosomal variation at high resolution continues to be a promising area of research that is yielding critical data regarding the genetic underpinnings of childhood psychiatric disorders. Preliminary data indicate that apparently identical submicroscopic variations in chromosomal structure may predispose to a very broad range of phenotypes. These findings suggest that disruption of the same basic neurodevelopmental mechanisms, such as synapse function, may result in outcomes that span a broad sweep of DSM-IV psychiatric diagnoses.

De novo damaging coding mutations are strongly associated with obsessive-compulsive disorder and overlap with autism

Obsessive-compulsive disorder (OCD) is a debilitating developmental neuropsychiatric disorder with a genetic risk component, yet identification of high-confidence risk genes has been challenging. We performed whole-exome sequencing in 222 OCD parent-child trios (184 trios after quality control), finding strong evidence that de novo likely gene disrupting and predicted damaging missense variants contribute to OCD risk. Together, these de novo damaging variants are enriched in OCD probands (RR 1.52, p=0.0005). We identified two high-confidence risk genes, each containing two de novo damaging variants in unrelated probands: CHD8 (Chromodomain Helicase DNA Binding Protein 8) and SCUBE1 (Signal Peptide, CUB Domain And EGF Like Domain Containing 1). Based on our data, we estimate that 34% of de novo damaging variants seen in OCD contribute to risk, and that de novo damaging variants in approximately 335 genes contribute to risk in 22% of OCD cases. Furthermore, genes harboring de novo damaging variants in OCD are enriched for those reported in neurodevelopmental disorders, particularly autism spectrum disorders. An exploratory network analysis reveals significant functional connectivity and enrichment in canonical pathways related to immune response. SIGNIFICANCE STATEMENT Decades of genetic studies in obsessive-compulsive disorder (OCD) have yet to provide reproducible, statistically significant findings. Following an approach that has led to tremendous success in gene discovery for several neuropsychiatric disorders, here we report findings from DNA whole-exome sequencing of patients with OCD and their parents. We find strong evidence for the contribution of spontaneous, or de novo, predicted-damaging genetic variants to OCD risk, identify two high-confidence risk genes, and detect significant overlap with genes previously identified in autism. These results change the status quo of OCD genetics by identifying novel OCD risk genes, clarifying the genetic landscape of OCD with respect to de novo variation, and suggesting underlying biological pathways that will improve our understanding of OCD biology.

Zebrafish: A Translational Model System for Studying Neuropsychiatric Disorders.

ith the advent of next-generation sequencing technologies, there has been considerable W progress in our ability to identify genes that are strongly associated with neurodevelopmental disorders, particularly autism spectrum disorders (ASDs). Despite the challenges of clinical and genetic heterogeneity, which complicate the identification of susceptibility genes, wholeexome sequencing of large cohorts of affected individuals has led to a rapidly expanding list of reliable risk genes that are beginning to illuminate basic neurobiological processes involved in ASDs, such as synaptic dysfunction. Moreover, the application of emerging genomic technologies to other neuropsychiatric disorders, such as schizophrenia, is beginning to provide deeper insights into the pathophysiology of these disorders. However, we now face the challenge of translating findings from large-scale genetics studies into a mechanistic understanding of neuropsychiatric disorders as a prerequisite for developing improved, target-driven pharmacotherapies. The zebrafish is emerging as a highly tractable model system to address this challenge. The growing popularity of this system is due in large part to key advantages of zebrafish over more traditional preclinical models. For example, zebrafish have rapid, external development of transparent embryos, which enables the visualization of basic neurobiological processes, such as neuronal migration and axon outgrowth, in real time in a living organism. In this way, zebrafish offer an invaluable window into the cell types and developmental stages that are affected when a risk gene is not functioning properly. Moreover, zebrafish larvae are small, easy to handle, and display a range of simple, quantifiable locomotor behaviors that provide a readout of circuit-level activity in a living, behaving organism. Because zebrafish may have progenies containing hundreds of embryos, such behavioral assays can be harnessed as a platform for conducting high-throughput small molecule screens and identifying pharmacological candidates that affect a simple behavior or circuit. Finally, the recent availability of genetic technologies for the rapid and cost-effective generation of zebrafish “knockouts,” in which the function of a gene of interest has been disrupted, will enable the application of the zebrafish model to a range of neuropsychiatric disorders (Figure 1A). Given these advantages, zebrafish have the potential to emerge as a key player in the path from risk gene discovery to the development of mechanism-based pharmacological interventions (Figure 1).

Zebrafish Models of Neurodevelopmental Disorders: Past, Present, and Future

Zebrafish are increasingly being utilized as a model system to investigate the function of the growing list of risk genes associated with neurodevelopmental disorders. This is due in large part to the unique features of zebrafish that make them an optimal system for this purpose, including rapid, external development of transparent embryos, which enable the direct visualization of the developing nervous system during early stages, large progenies, which provide considerable tractability for performing high-throughput pharmacological screens to identify small molecule suppressors of simple behavioral phenotypes, and ease of genetic manipulation, which has been greatly facilitated by the advent of CRISPR/Cas9 gene editing technologies. This review article focuses on studies that have harnessed these advantages of the zebrafish system for the functional analysis of genes that are strongly associated with the following neurodevelopmental disorders: autism spectrum disorders (ASD), epilepsy, intellectual disability (ID) and schizophrenia. We focus primarily on studies describing early morphological and behavioral phenotypes during embryonic and larval stages resulting from loss of risk gene function. We highlight insights into basic mechanisms of risk gene function gained from these studies as well as limitations of studies to date. Finally, we discuss advances in in vivo neural circuit imaging in zebrafish, which promise to transform research using the zebrafish model by illuminating novel circuit-level mechanisms with relevance to neurodevelopmental disorders.