Impaired Neurodevelopmental Genes in Slovenian Autistic Children Elucidate the Comorbidity of Autism With Other Developmental Disorders.

Autism spectrum disorders (ASD) represent a phenotypically heterogeneous group of patients that strongly intertwine with other neurodevelopmental disorders (NDDs), with genetics playing a significant role in their etiology. Whole exome sequencing (WES) has become predominant in molecular diagnostics for ASD by considerably increasing the diagnostic yield. However, the proportion of undiagnosed patients still remains high due to complex clinical presentation, reduced penetrance, and lack of segregation analysis or clinical information. Thus, reverse phenotyping, where we first identified a possible genetic cause and then determine its clinical relevance, has been shown to be a more efficient approach. WES was performed on 147 Slovenian pediatric patients with suspected ASD. Data analysis was focused on identifying ultrarare or "single event" variants in ASD-associated genes and further expanded to NDD-associated genes. Protein function and gene prioritization were performed on detected clinically relevant variants to determine their role in ASD etiology and phenotype. Reverse phenotyping revealed a pathogenic or likely pathogenic variant in ASD-associated genes in 20.4% of patients, with subsequent segregation analysis indicating that 14 were de novo variants and 1 was presumed compound heterozygous. The diagnostic yield was further increased by 2.7% by the analysis of ultrarare or "single event" variants in all NDD-associated genes. Protein function analysis established that genes in which variants of unknown significance (VUS) were detected were predominantly the cause of intellectual disability (ID), and in most cases, features of ASD as well. Using such an approach, variants in rarely described ASD-associated genes, such as SIN3B, NR4A2, and GRIA1, were detected. By expanding the analysis to include functionally similar NDD genes, variants in KCNK9, GNE, and other genes were identified. These would probably have been missed by classic genotype-phenotype analysis. Our study thus demonstrates that in patients with ASD, analysis of ultrarare or "single event" variants obtained using WES with the inclusion of functionally similar genes and reverse phenotyping obtained a higher diagnostic yield despite limited clinical data. The present study also demonstrates that most of the causative genes in our cohort were involved in the syndromic form of ASD and confirms their comorbidity with other developmental disorders.

Orphan Nuclear Receptor NR4A2 Is Constitutively Expressed in Cartilage and Upregulated in Inflamed Synovium From hTNF-Alpha Transgenic Mice.

Orphan nuclear receptor 4A2 (NR4A2/Nurr1) is a constitutively active transcription factor with potential roles in the onset and progression of inflammatory arthropathies. NR4A2 is overexpressed in synovium and cartilage from individuals with rheumatoid arthritis (RA), psoriatic arthritis, and osteoarthritis. This study documents the expression and tissue localization of NR4A2 and upstream regulator nuclear factor kappa B (NF-κB) in the human tumor necrosis factor-alpha (hTNF-α) transgenic mouse model of RA. Since TNF-α is a potent inducer of NR4A2 in vitro, we hypothesized that NR4A2 would also be upregulated and active during disease progression in this model. Expression levels of NR4A2, related receptors NR4A1 (Nur77) and 3 (NOR1), and NF-κB1 transcripts were quantified by RT-qPCR in hTNF-α and wild-type joints at three stages of disease. The protein distribution of NR4A2 and NF-κB subunit RelA (p65) was analyzed by quantitative immunohistochemistry. Global gene expression of 88 RA-related genes was also screened and compared between groups. Consistent with previous reports on the hTNF-α model, transgenic mice exhibited significant weight loss and severely swollen paws by 19 weeks of age compared to age-matched wild-type controls. NR4A1-3 and NF-κB1 were constitutively expressed at disease onset and in healthy joints. NF-κB1 transcript levels increased 2-fold in hTNF-α paws with established disease (12 weeks), followed by a 2-fold increase in NR4A2 at the late disease stage (19 weeks). NR4A2 and RelA proteins were overexpressed in inflamed synovium prior to symptoms of arthritis, suggesting that gene expression changes documented in whole paws were largely driven by elevated expression in diseased synovium. Broader screening of RA-related genes by RT-qPCR identified several differentially expressed genes in hTNF-α joints including those encoding inflammatory cytokines and chemokines, matrix-degrading enzymes and inhibitors, cell surface receptors, intracellular signaling proteins and transcription factors. Consensus binding sites for NR4A receptors and NF-κB1 were enriched in the promoters of differentially expressed genes suggesting central roles for these transcription factors in this model. This study is the first comprehensive analysis of NR4A2 in an animal model of RA and validates the hTNF-α model for testing of small molecules and genetic strategies targeting this transcription factor.

microRNA-137 promotes endothelial progenitor cell proliferation and angiogenesis in cerebral ischemic stroke mice by targeting NR4A2 through the Notch pathway.

Cerebral ischemic stroke (CIS) is one of the common causes of death and disability worldwide. This study aims to investigate effect of miR-137 on endothelial progenitor cells and angiogenesis in CIS by targeting NR4A2 via the Notch pathway. Brain tissues were extracted from CIS and normal mice. Immunohistochemistry was used to determine positive rate of NR4A2 expression. Serum VEGF, Ang, HGF, and IκBα levels were determined by ELISA. RT-qPCR and Western blotting were used to determine expression of related factors. Endothelial progenitor cells in CIS mice were treated and grouped into blank, NC, miR-137 mimic, miR-137 inhibitor, siRNA-NR4A2, and miR-137 inhibitor + siRNA-NR4A2 groups, and cells in normal mice into normal group. Proliferation and apoptosis were determined by MTT and flow cytometry, respectively. NR4A2 protein expression was strongly positive in CIS mice, which showed higher serum levels of VEGF, Ang, and HGF but lower IκBα than normal mice. Compared with normal group, the rest groups (endothelial progenitor cells from CIS mice) showed decreased expressions of miR-137, Hes1, Hes5, and IκBα but elevated NR4A2, Notch, Jagged1, Hey-2, VEGF, Ang, and HGF, inhibited proliferation and enhanced apoptosis. Compared with blank and NC groups, the miR-137 mimic and siRNA-NR4A2 groups exhibited increased expression of miR-137, Hes1, Hes5, and IκBα, but decreased NR4A2, Notch, Jagged1, and Hey-2, with enhanced proliferation and attenuated apoptosis. The miR-137 inhibitor group reversed the conditions. miR-137 enhances the endothelial progenitor cell proliferation and angiogenesis in CIS mice by targeting NR4A2 through the Notch signaling pathway.

Association of polymorphisms and reduced expression levels of the NR4A2 gene with Parkinson's disease in a Mexican population.

INTRODUCTION: The NR4A2 transcription factor is important in the development, survival and phenotype of dopaminergic neurons and it is postulated as a possible biomarker for Parkinson's disease (PD). Therefore, our aim was to analyze in a sample of a Mexican population with idiopathic PD, mutations (in two hotspot mutation regions) and two polymorphisms (rs34884856 in promotor and rs35479735 intronic regions) of the NR4A2 gene. We also evaluate the levels of NR4A2 gene expression in peripheral blood for a Mexican population, and identify whether they are associated with NR4A2 gene polymorphisms. METHODS: We conducted a case-control study, which included 227 idiopathic PD cases and 454 unrelated controls. Genetic variants of the NR4A2 gene were genotyped by high-resolution melting (HRM) and validated by an automated sequencing method. The gene expression was performed in peripheral blood using a real-time polymerase chain reaction. RESULTS: The rs35479735 polymorphism was associated with a higher risk of developing PD. In addition, NR4A2 gene expression was significantly decreased in patients with PD. Linkage disequilibrium analysis showed a haplotype H4 (3C-3G) that showed lower levels of expression, and contained the risk alleles for both polymorphisms. CONCLUSIONS: In summary, this is the first study in a Mexican population that considers the analysis of NR4A2 in patients with PD. An association was identified between genotype and mRNA expression levels of NR4A2 in patients with PD. These results suggest that polymorphisms and expression of the NR4A2 gene could play an important role in the risk of developing PD in Mexican populations.