Association of NEF2L2 Rs35652124 Polymorphism with Nrf2 Induction and Genotoxic Stress Biomarkers in Autism.

Increased oxidative/genotoxic stress is known to impact the pathophysiology of ASD (autism spectrum disorder). Clinical studies, however, reported limited, heterogeneous but promising responses to treatment with antioxidant remedies. We determined whether the functional polymorphism of the Nrf2 gene, master regulator of anti-oxidant adaptive reactions to genotoxic stress, links to the genotoxic stress responses and to an in vitro effect of a NRF2 inductor in ASD children. Oxidative stress biomarkers, adaptive responses to genotoxic/oxidative stress, levels of master antioxidant regulator Nrf2 and its active form pNrf2 before and after inducing by dimethyl fumarate (DMF), and promotor rs35652124 polymorphism of NFE2L2 gene encoding Nrf2 were studied in children with ASD (n = 179). Controls included healthy adults (n = 101). Adaptive responses to genotoxicity as indicated by H2AX and cytoprotection by NRF2 contents positively correlated in ASD children with a Spearman coefficient of R = 0.479 in T+, but not CC genotypes. ASD children with NRF2 rs35652124 CC genotype demonstrated significantly higher H2AX content (0.652 vs. 0.499 in T+) and pNrf2 induction by DMF, lowered 8-oxo-dG concentration in plasma and higher cfDNA/plasma nuclease activity ratio. Our pilot findings suggest that in ASD children the NEF2L2 rs35652124 polymorphism impacts adaptive responses that may potentially link to ASD severity. Our data warrant further studies to reveal the potential for NEF2L2 genotype-specific and age-dependent repurposing of DMF and/or other NRF2-inducing drugs.

Ribosomal DNA Abundance in the Patient's Genome as a Feasible Marker in Differential Diagnostics of Autism and Childhood-Onset Schizophrenia.

Introduction: Differential diagnostics of early-onset schizophrenia and autism spectrum disorders (ASD) are a problem of child psychiatry. The prognosis and relevant treatment are to a large degree determined by the correctness of diagnosis. We found earlier that leucocyte DNA of adult schizophrenia patients contained significantly larger copy numbers of ribosomal repeats (rDNA) coding for rRNA, than DNA of mentally healthy controls. Aim: To compare the contents of ribosomal repeats in the leucocyte DNA of children with schizophrenia, children with ASD, and healthy age-matched controls to estimate the possibility of using this genetic trait in the differential diagnostics of the two types of disorders. Patients and methods: Blood samples of patients with infantile autism (A­F84.0 according to ICD-10, N = 75) and with childhood-onset schizophrenia (SZ­F20.8 according to ICD-10, N = 43) were obtained from the Child Psychiatry Department of the Mental Health Research Center. The healthy control blood samples (HC, N = 86) were taken from the Research Centre for Medical Genetics collection. The recruitment of cases was based on the clinical psychopathologic approach. DNA was extracted from blood leukocytes with organic solvents. Nonradioactive quantitative hybridization technique was applied for determining the abundance of ribosomal repeats in the genomes. Statistical processing was performed using StatPlus, Statgraphics and MedCalc. Findings: DNA derived from SZ cases contained 565 ± 163 rDNA copies, which is significantly (p < 10−6) higher than the rDNA content in ASD cases (405 ± 109 copies) and controls (403 ± 86 copies). The HC and A groups did not differ by rDNA copy number (p > 0.4). The genetic trait "rDNA copy number in patient's genome" can potentially be applied as an additional marker in differential diagnostics of childhood-onset schizophrenia and autism spectrum disorders.

Oxidized cell-free DNA as a stress-signaling factor activating the chronic inflammatory process in patients with autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASD) are known to be associated with an inflammatory process related to immune system dysfunction. This study's aim was to investigate the role of cell-free DNA in chronic inflammatory process in ASD patients. METHODS: The study included 133 ASD patients and 27 healthy controls. Sixty-two ASD patients were demonstrated to have mild-to-moderate disease severity (group I) and 71 individuals to have severe ASD (group II). Plasma cell-free (cf) DNA characteristics, plasma cytokine concentrations, expression of the genes for NFкB1 transcription factor and pro-inflammatory cytokines TNFα, IL-1ß and IL-8 in peripheral blood lymphocytes (PBL) of ASD patients, and unaffected controls were investigated. Additionally, in vitro experiments with oxidized DNA supplementation to PBL cultures derived from ASD patients and healthy controls were performed. RESULTS: The data indicates that ASD patients have demonstrated increased cfDNA concentration in their circulation. cfDNA of patients with severe ASD has been characterized by a high abundance of oxidative modification. Furthermore, ASD patients of both groups have shown elevated plasma cytokine (IL-1ß, IL-8, IL-17A) levels and heightened expression of genes for NFкB1 nuclear factor and pro-inflammatory cytokines TNFα, IL-1ß, and IL-8 in PBL. In vitro experiments have shown that NF-κB/cytokine mRNA expression profiles of ASD patient PBL treated with oxidized DNA fragments were significantly different from those of healthy controls. CONCLUSIONS: It may be proposed that oxidized cfDNA plays a role of stress-signaling factor activating the chronic inflammatory process in patients with ASD.

Changes of KEAP1/NRF2 and IKB/NF-κB Expression Levels Induced by Cell-Free DNA in Different Cell Types.

Cell-free DNA (cfDNA) is a circulating DNA of nuclear and mitochondrial origin mainly derived from dying cells. Recent studies have shown that cfDNA is a stress signaling DAMP (damage-associated molecular pattern) molecule. We report here that the expression profiles of cfDNA-induced factors NRF2 and NF-κB are distinct depending on the target cell's type and the GC-content and oxidation rate of the cfDNA. Stem cells (MSC) have shown higher expression of NRF2 without inflammation in response to cfDNA. In contrast, inflammatory response launched by NF-κB was dominant in differentiated cells HUVEC, MCF7, and fibroblasts, with a possibility of transition to massive apoptosis. In each cell type examined, the response for oxidized cfDNA was more acute with higher peak intensity and faster resolution than that for nonoxidized cfDNA. GC-rich nonoxidized cfDNA evoked a weaker and prolonged response with proinflammatory component (NF-κB) as predominant. The exploration of apoptosis rates after adding cfDNA showed that cfDNA with moderately increased GC-content and lightly oxidized DNA promoted cell survival in a hormetic manner. Novel potential therapeutic approaches are proposed, which depend on the current cfDNA content: either preconditioning with low doses of cfDNA before a planned adverse impact or eliminating (binding, etc.) cfDNA when its content has already become high.