Combination of machine learning-based bulk and single-cell genomics reveals necroptosis-related molecular subtypes and immunological features in autism spectrum disorder.

Background: Necroptosis is a novel form of controlled cell death that contributes to the progression of various illnesses. Nonetheless, the function and significance of necroptosis in autism spectrum disorders (ASD) remain unknown and require further investigation. Methods: We utilized single-nucleus RNA sequencing (snRNA-seq) data to assess the expression patterns of necroptosis in children with autism spectrum disorder (ASD) based on 159 necroptosis-related genes. We identified differentially expressed NRGs and used an unsupervised clustering approach to divide ASD children into distinct molecular subgroups. We also evaluated immunological infiltrations and immune checkpoints using the CIBERSORT algorithm. Characteristic NRGs, identified by the LASSO, RF, and SVM-RFE algorithms, were utilized to construct a risk model. Moreover, functional enrichment, immune infiltration, and CMap analysis were further explored. Additionally, external validation was performed using RT-PCR analysis. Results: Both snRNA-seq and bulk transcriptome data demonstrated a greater necroptosis score in ASD children. Among these cell subtypes, excitatory neurons, inhibitory neurons, and endothelials displayed the highest activity of necroptosis. Children with ASD were categorized into two subtypes of necroptosis, and subtype2 exhibited higher immune activity. Four characteristic NRGs (TICAM1, CASP1, CAPN1, and CHMP4A) identified using three machine learning algorithms could predict the onset of ASD. Nomograms, calibration curves, and decision curve analysis (DCA) based on 3-NRG have been shown to have clinical benefit in children with ASD. Furthermore, necroptosis-based riskScore was found to be positively associated with immune activation. Finally, RT-PCR demonstrated differentially expressed of these four NRGs in human peripheral blood samples. Conclusion: A comprehensive identification of necroptosis may shed light on the underlying pathogenic process driving ASD onset. The classification of necroptosis subtypes and construction of a necroptosis-related risk model may yield significant insights for the individualized treatment of children with ASD.