Maternal stress in Shank3ex4-9 mice increases pup-directed care and alters brain white matter in male offspring.

Gene-environment interactions contribute to the risk for Autism Spectrum Disorder (ASD). Among environmental factors, prenatal exposure to stress may increase the risk for ASD. To examine if there is an interaction between exposure to maternal stress and reduced dosage or loss of Shank3, wild-type (WT), heterozygous (HET) and homozygous (HOM) female mice carrying a deletion of exons four through nine of Shank3 (Shank3ex4-9) were exposed to chronic unpredictable mild stress (CUMS) from prior to conception throughout gestation. This study examined maternal care of these dams and the white matter microstructure in the brains of their adult male offspring. Overall, our findings suggest that maternal exposure to CUMS increased pup-directed care for dams of all three genotypes. Compared to WT and HET dams, HOM dams also exhibited increased maternal care behaviors with increased time spent in the nest and reduced cage exploration, regardless of exposure to CUMS. Diffusion tensor imaging showed higher mean fractional anisotropy in the hippocampal stratum radiatum of WT and HOM male offspring from dams exposed to CUMS and HOM offspring from unexposed dams, compared to WT male offspring from unexposed dams. These data support that CUMS in Shank3-mutant dams results in subtle maternal care alterations and long-lasting changes in the white matter of the hippocampus of their offspring.

Phenotypic characterization of individuals with SYNGAP1 pathogenic variants reveals a potential correlation between posterior dominant rhythm and developmental progression.

BACKGROUND: The SYNGAP1 gene encodes for a small GTPase-regulating protein critical to dendritic spine maturation and synaptic plasticity. Mutations have recently been identified to cause a breadth of neurodevelopmental disorders including autism, intellectual disability, and epilepsy. The purpose of this work is to define the phenotypic spectrum of SYNGAP1 gene mutations and identify potential biomarkers of clinical severity and developmental progression. METHODS: A retrospective clinical data analysis of individuals with SYNGAP1 mutations was conducted. Data included genetic diagnosis, clinical history and examinations, neurophysiologic data, neuroimaging, and serial neurodevelopmental/behavioral assessments. All patients were seen longitudinally within a 6-year period; data analysis was completed on June 30, 2018. Records for all individuals diagnosed with deleterious SYNGAP1 variants (by clinical sequencing or exome sequencing panels) were reviewed. RESULTS: Fifteen individuals (53% male) with seventeen unique SYNGAP1 mutations are reported. Mean age at genetic diagnosis was 65.9 months (28-174 months). All individuals had epilepsy, with atypical absence seizures being the most common semiology (60%). EEG abnormalities included intermittent rhythmic delta activity (60%), slow or absent posterior dominant rhythm (87%), and epileptiform activity (93%), with generalized discharges being more common than focal. Neuroimaging revealed nonspecific abnormalities (53%). Neurodevelopmental evaluation revealed impairment in all individuals, with gross motor function being the least affected. Autism spectrum disorder was diagnosed in 73% and aggression in 60% of cases. Analysis of biomarkers revealed a trend toward a moderate positive correlation between visual-perceptual/fine motor/adaptive skills and language development, with posterior dominant rhythm on electroencephalogram (EEG), independent of age. No other neurophysiology-development associations or correlations were identified. CONCLUSIONS: A broad spectrum of neurologic and neurodevelopmental features are found with pathogenic variants of SYNGAP1. An abnormal posterior dominant rhythm on EEG correlated with abnormal developmental progression, providing a possible prognostic biomarker.

Global Health Education in Child Neurology and Neurodevelopmental Disabilities Training Programs in the United States: A National Survey.

OBJECTIVES: To describe the current landscape of opportunities and education in global health among child neurology and neurodevelopmental disabilities training programs and provide a framework for future development of global health education. METHODS: Authors surveyed Trainee and Program Director groups, obtaining information regarding global health interest, participation and obstacles (trainees); and collaborations in global health, academic yield and obstacles, and global health educational development within the training program (program directors). RESULTS: Of identified trainees and program directors, 35% and 48% responded, respectively. Among trainees, 82% reported interest in global health, with 25% reporting influence in program selection. Among program directors, 34% reported global health collaborations, most frequently clinical. Academic yield (conference participation or publications) was described by 46% of programs. Major obstacles described by both groups included administrative issues and funding; however, the latter was most important for program directors but not for trainees. Among program directors, 16% reported global health curricula, with lectures (100%), orientation courses (50%), and pre/post-travel sessions (50%) being commonest elements. The main content included education in public health, resourcefulness, and epidemiology. Half of responding programs offered a formal global health training track, including opportunities in language education (67%) and advanced degrees (33%). CONCLUSIONS: Similar to other specialties, growing interest in global health among trainees corresponds to growing availability of said opportunities; however, most display significant logistic obstacles and lack curricular development. Potential areas for intervention, including an interdisciplinary approach, and potential benefits to stakeholders are identified for programs wishing to expand in global health education.

Correction: Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring.

[This corrects the article DOI: 10.1371/journal.pone.0170127.].

Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring.

Offspring of murine dams chronically fed a protein-restricted diet have an increased risk for metabolic and neurobehavioral disorders. Previously we showed that adult offspring, developmentally exposed to a chronic maternal low-protein (MLP) diet, had lower body and hind-leg muscle weights and decreased liver enzyme serum levels. We conducted energy expenditure, neurobehavioral and circadian rhythm assays in male offspring to examine mechanisms for the body-weight phenotype and assess neurodevelopmental implications of MLP exposure. C57BL/6J dams were fed a protein restricted (8%protein, MLP) or a control protein (20% protein, C) diet from four weeks before mating until weaning of offspring. Male offspring were weaned to standard rodent diet (20% protein) and single-housed until 8-12 weeks of age. We examined body composition, food intake, energy expenditure, spontaneous rearing activity and sleep patterns and performed behavioral assays for anxiety (open field activity, elevated plus maze [EPM], light/dark exploration), depression (tail suspension and forced swim test), sociability (three-chamber), repetitive (marble burying), learning and memory (fear conditioning), and circadian behavior (wheel-running activity during light-dark and constant dark cycles). We also measured circadian gene expression in hypothalamus and liver at different Zeitgeber times (ZT). Male offspring from separate MLP exposed dams had significantly greater body fat (P = 0.03), less energy expenditure (P = 0.004), less rearing activity (P = 0.04) and a greater number of night-time rest/sleep bouts (P = 0.03) compared to control. MLP offspring displayed greater anxiety-like behavior in the EPM (P<0.01) but had no learning and memory deficit in fear-conditioning assay (P = 0.02). There was an effect of time on Per1, Per 2 and Clock circadian gene expression in the hypothalamus but not on circadian behavior. Thus, transplacental and early developmental exposure of dams to chronic MLP reduces food intake and energy expenditure, increases anxiety like behavior and disturbs sleep patterns but not circadian rhythm in adult male offspring.

No evidence for mutations in NLRP7 and KHDC3L in women with androgenetic hydatidiform moles.

OBJECTIVE: The objective of this study was to evaluate the mutational spectrum of NLRP7 and KHDC3L (C6orf221) in women with sporadic and recurrent androgenetic complete hydatidiform moles (AnCHM) and biparental hydatidiform moles (BiHM) to address the hypothesis that autosomal recessive mutations in these genes are only or primarily associated with BiHM. METHOD: We recruited 16 women with suspected recurrent and sporadic AnCHM and five women with suspected BiHM in addition to their reproductive partners into our study. We then sequenced the coding exons of NLRP7 and KHDC3L from DNA isolated from either blood or saliva from the study subjects. RESULTS: Sequence analysis of NLRP7 and KHDC3L revealed previously described single nucleotide polymorphisms in patients with AnCHM. However, in patients with BiHM, we identified a novel homozygous mutation and a previously described intragenic duplication of exons 2 to 5 in NLRP7, both of which are likely to be disease causing. We did not identify mutations in KHDC3L in patients with either form of hydatidiform moles. CONCLUSIONS: The absence of mutations in women with AnCHM supports a role for NLRP7 or KHDC3L in BiHM only. The absence of mutations in KHDC3L in women with BiHM is consistent with its minor role in this disease compared with NLRP7, the major BiHM gene.

Deletion of Porcn in mice leads to multiple developmental defects and models human focal dermal hypoplasia (Goltz syndrome).

BACKGROUND: Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated. RESULTS: We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion. CONCLUSIONS: These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.

Chronic maternal protein deprivation in mice is associated with overexpression of the cohesin-mediator complex in liver of their offspring.

Epigenetic mechanisms may play an important role in the developmental programming of adult-onset chronic metabolic diseases resulting from suboptimal fetal nutrition, but the exact molecular mechanisms are incompletely understood. Given the central role of the liver in metabolic regulation, we investigated whether chronic maternal dietary protein restriction has long-term effects on liver gene expression in the offspring. We fed adult C57BL/6J dams ad libitum an 8% maternal low-protein (MLP) or 20% protein control diet (C) from 4 wk prior to mating until the end of lactation. Male pups were weaned to standard nonpurified diet and singly housed at 21 d of age (d 21). Body weights were followed to 1 y of age (1 y). At d 21 and 1 y, organs were quantitatively dissected and analyzed. MLP offspring had significantly lower body weights at all ages and significantly lower serum activity of alanine aminotransferase and lactate dehydrogenase at 1 y. Gene expression profiling of liver at 1 y showed 521 overexpressed and 236 underexpressed genes in MLP compared to C offspring. The most important novel finding was the overexpression of genes found in liver that participate in organization and maintenance of higher order chromatin architecture and regulation of transcriptional activation. These included members of the cohesin-mediator complex, which regulate gene expression by forming DNA loops between promoters and enhancers in a cell type-specific fashion. Thus, our findings of increased expression of these factors in liver of MLP offspring implicate a possible novel epigenetic mechanism in developmental programming.

Colonic mucosal DNA methylation, immune response, and microbiome patterns in Toll-like receptor 2-knockout mice.

The connection between intestinal microbiota and host physiology is increasingly becoming recognized. The details of this dynamic interaction, however, remain to be explored. Toll-like receptor 2 (Tlr2) is important for its role in bacterial recognition, intestinal inflammation, and obesity-related metabolic changes. Therefore, we sought to determine the epigenomic and metagenomic consequences of Tlr2 deficiency in the colonic mucosa of mice to gain insights into biological pathways that shape the interface between the gut microbiota and the mammalian host. Colonic mucosa from wild type (WT) and Tlr2(-/-) C57BL/6 mice was interrogated by microarrays specific for DNA methylation and gene expression. The mucosal microbiome was studied by next-generation pyrosequencing of bacterial 16S rRNA. The expression of genes involved in immune processes was significantly modified by the absence of Tlr2, a number of which correlated with DNA methylation changes. The epigenomic and transcriptomic modifications associated with alteration in mucosal microbial composition. Several bacterial species, including members of the Firmicutes were significantly different in abundance between WT and Tlr2(-/-) animals. This manuscript highlights the intimate interrelationships between expression of immune-related genes and immunity pathways in the host with compositional and functional differences of the mammalian microbiome.

Epigenetic maturation in colonic mucosa continues beyond infancy in mice.

Monozygotic twin and other epidemiologic studies indicate that epigenetic processes may play an important role in the pathogenesis of inflammatory bowel diseases that commonly affect the colonic mucosa. The peak onset of these disorders in young adulthood suggests that epigenetic changes normally occurring in the colonic mucosa shortly before adulthood could be important etiologic factors. We assessed developmental changes in colitis susceptibility during the physiologically relevant period of childhood in mice [postnatal day 30 (P30) to P90] and concurrent changes in DNA methylation and gene expression in murine colonic mucosa. Susceptibility to colitis was tested in C57BL/6J mice with the dextran sulfate sodium colitis model. Methylation specific amplification microarray (MSAM) was used to screen for changes in DNA methylation, with validation by bisulfite pyrosequencing. Gene expression changes were analyzed by microarray expression profiling and real time RT-PCR. Mice were more susceptible to chemically induced colitis at P90 than at P30. DNA methylation changes, however, were not extensive; of 23 743 genomic intervals interrogated, only 271 underwent significant methylation alteration during this developmental period. We found an excellent correlation between the MSAM and bisulfite pyrosequencing at 11 gene associated intervals validated (R(2) = 0.89). Importantly, at the genes encoding galectin-1 (Lgals1), and mothers against decapentaplegic homolog 3 or Smad3, both previously implicated in murine colitis, developmental changes in DNA methylation from P30 to P90 were inversely correlated with expression. Colonic mucosal epigenetic maturation continues through early adulthood in the mouse, and may contribute to the age-associated increase in colitis susceptibility. Transcript Profiling: Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), accession numbers: GSE18031 (DNA methylation arrays), GSE19506 (gene expression arrays).

Assessment of DNA methylation at the interferon regulatory factor 5 (IRF5) promoter region in inflammatory bowel diseases.

BACKGROUND AND AIMS: A 5-bp insertion-deletion (indel) polymorphism in the promoter of interferon regulatory factor 5 (IRF5) has been associated with inflammatory bowel diseases (IBD). This polymorphism generates an additional binding site for the transcription factor SP1 and has been shown to augment the expression of IRF5. Additionally, it affects a CpG dinucleotide-dense genomic region. These features of the indel suggested that it may influence the epigenetic regulation of IRF5. The aim of this study was to investigate the potential effect of the 5-bp indel on the methylation pattern of four CpG sites upstream of the polymorphism. Possible CpG site methylation differences in this region between healthy persons and individuals suffering from IBD were also tested. METHODS: Genotype was determined by 4% polyacrylamide gel electrophoresis in 33 peripheral blood leukocyte (PBL) DNA samples. DNA methylation correlates of the genotypes were measured by bisulfite pyrosequencing. IRF5 promoter methylation in association to disease state was assessed in 87 proband (49 healthy, 18 Crohn's disease, 20 ulcerative colitis) PBL samples. RESULTS: The polymorphism did not affect the methylation pattern of the IRF5 promoter nor could we detect significant differences in the average, low methylation of the locus between healthy persons and individuals with IBD. CONCLUSIONS: These results implicate that epigenetic dysregulation of the IRF5 promoter is unlikely to be associated with IBD.

O-GlcNAc modification of proteins affects volume regulation in Jurkat cells.

An increasing amount of recent research has demonstrated that the hexosamine biosynthesis pathway (HBP) plays a significant role in the modulation of intracellular signaling transduction pathways, and affects cellular processes via modification of protein by O-linked beta-N-acetylglucosamine (O-GlcNAc). Besides the many known and postulated effects of protein O-GlcNAc modifications, there is little available data on the role of O-GlcNAc in cellular volume regulation. Our objective was to test the effect of increased O-GlcNAc levels on hypotonia-induced volume changes in Jurkat cells. We pretreated Jurkat cells for 1 h with glucosamine (GlcN), PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate) an inhibitor of O-GlcNAcase, or a high level of glucose to induce elevated levels of O-GlcNAc. We found that the response of Jurkat cells to hypotonic stress was significantly altered. The hypotonia induced cell-swelling was augmented in both GlcN and PUGNAc-treated cells and, to a lesser extent, in high glucose concentration-treated cells. Evaluated by NMR measurements, GlcN and PUGNAc treatment also significantly reduced intracellular water diffusion. Taken together, increased cell swelling and reduced water diffusion caused by elevated O-GlcNAc show notable analogy to the regulatory volume changes seen by magnetic resonance methods in nervous and other tissues in different pathological states. In conclusion, we demonstrate for the first time that protein O-GlcNAc could modulate cell volume regulation.