Artificial neural networks reveal sex differences in gene methylation, and connections between maternal risk factors and symptom severity in autism spectrum disorder.

Aim and methods: Artificial neural networks were used to unravel connections among blood gene methylation levels, sex, maternal risk factors and symptom severity evaluated using the Autism Diagnostic Observation Schedule 2 (ADOS-2) score in 58 children with autism spectrum disorder (ASD). Results: Methylation levels of MECP2, HTR1A and OXTR genes were connected to females, and those of EN2, BCL2 and RELN genes to males. High gestational weight gain, lack of folic acid supplements, advanced maternal age, preterm birth, low birthweight and living in rural context were the best predictors of a high ADOS-2 score. Conclusion: Artificial neural networks revealed links among ASD maternal risk factors, symptom severity, gene methylation levels and sex differences in methylation that warrant further investigation in ASD.

Vitamin D Status in Children with Autism Spectrum Disorders: Determinants and Effects of the Response to Probiotic Supplementation.

A relationship between the presence of clinical symptoms and gastrointestinal (GI) disturbances associated with nutritional deficiencies, including vitamin D (25(OH)D) deficiency, has been observed in autism spectrum disorder (ASD). The aim was to evaluate 25(OH)D levels according to the annual rhythm cycle, gender, the severity of autism, nutritional or clinical status, inflammatory and metabolic biomarkers, GI symptoms, and the clinical response to probiotic/placebo supplementation in preschooler children with ASD. Eighty-one ASD preschoolers (67 males) were assessed with standardized tools for ASD severity (ADOS score) and GI symptoms (by GI-Index at six-items and at nine-items, the latter defined as the Total GI-Index). The 25(OH)D levels were compared among different ASD subgroups according to metabolic and inflammatory biomarkers (leptin, insulin, resistin, PAI-1, MCP-1, TNF-alfa, and IL-6), gender, and the presence or absence of: (i) GI symptoms, (ii) the response to probiotic supplementation (the improvement of GI symptomatology), (iii) the response to probiotic supplementation (improvement of ASD severity). Only 25% of the ASD children presented an adequate 25(OH)D status (≥30 ng/mL according to the Endocrine Society guidelines). All the 25(OH)D levels falling in the severe deficiency range (<10 ng/mL) were observed in the male subgroup. A significant inverse correlation between 25(OH)D and leptin was observed (R = −0.24, p = 0.037). An inverse correlation was found between 25(OH)D levels and the GI Index 6-Items and Total GI-Index (R = −0.25, p = 0.026; −0.27, = 0.009) and a direct relationship with the probiotic response (R = 0.4, p = 0.05). The monitoring of 25(OH)D levels and the co-administration of 25(OH)D and probiotic supplementation could be considered in ASD from early ages.

Correlation among maternal risk factors, gene methylation and disease severity in females with autism spectrum disorder.

Aim: To detect early-life environmental factors leading to DNA methylation changes of autism spectrum disorder (ASD)-related genes in young ASD females and reveal epigenetic biomarkers of disease severity. Materials & methods: We investigated blood methylation levels of MECP2, OXTR, BDNF, RELN, BCL2, EN2 and HTR1A genes in 42 ASD females. Results: Maternal gestational weight gain correlated with BDNF methylation levels (Bonferroni-corrected p = 0.034), and lack of folic acid supplementation at periconception resulted in higher disease severity in the ASD children (Bonferroni-corrected p = 0.048). RELN methylation levels were inversely correlated with disease severity (Bonferroni corrected p = 0.042). Conclusion: The present study revealed gene-environment interactions and potential epigenetic biomarkers of disease severity in ASD females.

Early-life maternal factors can leave marks on the DNA of the developing fetus, including changes in DNA methylation that regulate gene expression levels. These marks can pose an increased risk for several diseases, such as autism spectrum disorder (ASD) and other developmental disorders. In the present study, we searched for links between early-life maternal factors and the methylation levels of ASD-related genes in blood DNA samples of young ASD diagnosed females. We found that high maternal gestational weight gain resulted in increased methylation levels of the BDNF gene, one of the most important genes for brain development. Moreover, lack of maternal folic acid supplementation and low RELN methylation levels resulted in higher disease severity in ASD females.

Gut to brain interaction in Autism Spectrum Disorders: a randomized controlled trial on the role of probiotics on clinical, biochemical and neurophysiological parameters.

BACKGROUND: A high prevalence of a variety of gastrointestinal (GI) symptoms is frequently reported in patients with Autism Spectrum Disorders (ASD). The GI disturbances in ASD might be linked to gut dysbiosis representing the observable phenotype of a "gut-brain axis" disruption. The exploitation of strategies which can restore normal gut microbiota and reduce the gut production and absorption of toxins, such as probiotics addition/supplementation in a diet, may represent a non-pharmacological option in the treatment of GI disturbances in ASD. The aim of this randomized controlled trial is to determine the effects of supplementation with a probiotic mixture (Vivomixx®) in ASD children not only on specific GI symptoms, but also on the core deficits of the disorder, on cognitive and language development, and on brain function and connectivity. An ancillary aim is to evaluate possible effects of probiotic supplementation on urinary concentrations of phthalates (chemical pollutants) which have been previously linked to ASD. METHODS: A group of 100 preschoolers with ASD will be classified as belonging to a GI group or to a Non-GI (NGI) group on the basis of a symptom severity index specific to GI disorders. In order to obtain four arms, subjects belonging to the two groups (GI and NGI) will be blind randomized 1:1 to regular diet with probiotics or with placebo for 6 months. All participants will be assessed at baseline, after three months and after six months from baseline in order to evaluate the possible changes in: (1) GI symptoms; (2) autism symptoms severity; (3) affective and behavioral comorbid symptoms; (4) plasmatic, urinary and fecal biomarkers related to abnormal intestinal function; (5) neurophysiological patterns. DISCUSSION: The effects of treatments with probiotics on children with ASD need to be evaluated through rigorous controlled trials. Examining the impact of probiotics not only on clinical but also on neurophysiological patterns, the current trial sets out to provide new insights into the gut-brain connection in ASD patients. Moreover, results could add information to the relationship between phthalates levels, clinical features and neurophysiological patterns in ASD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02708901 . Retrospectively registered: March 4, 2016.

Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain.

The involvement of melatonin in mammalian brain pathophysiology has received growing interest, but information about the anatomical distribution of its two G-protein-coupled receptors, MT1 and MT2 , remains elusive. In this study, using specific antibodies, we examined the precise distribution of both melatonin receptors immunoreactivity across the adult rat brain using light, confocal, and electron microscopy. Our results demonstrate a selective MT1 and MT2 localization on neuronal cell bodies and dendrites in numerous regions of the rat telencephalon, diencephalon, and mesencephalon. Confocal and ultrastructural examination confirmed the somatodendritic nature of MT1 and MT2 receptors, both being localized on neuronal membranes. Overall, striking differences were observed in the anatomical distribution pattern of MT1 and MT2 proteins, and the labeling often appeared complementary in regions displaying both receptors. Somadendrites labeled for MT1 were observed for instance in the retrosplenial cortex, the dentate gyrus of the hippocampus, the islands of Calleja, the medial habenula, the suprachiasmatic nucleus, the superior colliculus, the substantia nigra pars compacta, the dorsal raphe nucleus, and the pars tuberalis of the pituitary gland. Somadendrites endowed with MT2 receptors were mostly observed in the CA3 field of the hippocampus, the reticular thalamic nucleus, the supraoptic nucleus, the inferior colliculus, the substantia nigra pars reticulata, and the ventrolateral periaqueductal gray. Together, these data provide the first detailed neurocytological mapping of melatonin receptors in the adult rat brain, an essential prerequisite for a better understanding of melatonin distinct receptor function and neurophysiology.