Clinical and laboratory criteria for intestinal barrier permeability impairment and gut microbiome alterations in children with autism spectrum disorder based on zonulin, calprotectin and 16S rRNA gene sequencing

Abstract

Introduction. The study of the intestinal barrier and microbiota is of interest for the comprehensive characterization of children with autism spectrum disorder (ASD). Objective. To characterize the clinical and laboratory features of the intestinal barrier and microbiota in children with ASD by assessing fecal zonulin and calprotectin levels and performing 16S rRNA gene sequencing. Materials and Methods. The study was conducted between 2023 and 2026 at the U. K. Kurbanov Republican Children’s Psychoneurological Hospital and the GenScreen laboratory (Tashkent). A total of 274 children aged 2 to 8 years (mean age 4.4 ± 0.6 years) were examined. The study group consisted of 169 patients with a confirmed ASD diagnosis (147 boys and 22 girls). The control group comprised 105 healthy children of similar age (69 boys and 36 girls). Clinical and behavioral status was assessed using standardized instruments: M-CHAT-R, CASD, and ADOS-2. Fecal zonulin and calprotectin levels were measured via enzyme-linked immunosorbent assay (ELISA) using commercial test kits at the PRIME specialized laboratory (Tashkent). For the molecular-genetic analysis of the gut microbiota structure, a subset of 27 patients with ASD underwent high-throughput next-generation sequencing (NGS) of the 16S rRNA gene. Bioinformatic processing included chimera filtering and the clustering of amplicon sequence variants (ASVs), with taxonomic annotation performed against the SILVA database. Statistical analysis was performed in the R software environment (v. 4.x) using the non-parametric Mann–Whitney U test, Fisher’s exact test, and Spearman’s rank correlation with the Benjamini–Hochberg (FDR) correction. Aim: To characterize the clinical and laboratory features of impaired intestinal barrier permeability and microbiota composition in children with ASD, based on the assessment of fecal zonulin, calprotectin, and 16S rRNA gene sequencing. Results and discussion: Eating disorders were identified in 40.8% of children with ASD, compared to 9.5% in the control group. Fecal zonulin levels in the study group were more than twice as high as those in the control group (84.5 ng/mL vs. 38.2 ng/mL; p < 0.001), indicating the onset of the degradation of tight junction proteins in the mucous membrane. Fecal calprotectin concentrations in children with ASD also showed a marked increase, reaching 78.3 µg/g, compared to 22.4 µg/g in the control group (p < 0.001). A significant positive correlation was found between zonulin levels and the total autism severity score on the ADOS-2 scale (ρ = +0.58; p_FDR < 0.001), as well as the severity of stereotypical behaviors (ρ = +0.49; p < 0.01). Within the microbiome profile, zonulin levels were positively associated with the abundance of Clostridium spp. (ρ = +0.61; p < 0.001) and *Bacteroides fragilis* (ρ = +0.54; p < 0.001). Calprotectin concentration showed a highly significant correlation with the presence of eating disorders (ρ = +0.59; p < 0.001) and the severity of affective outbursts in children (ρ = +0.47; p_FDR = 0.004). Conclusion. This study provides the first comprehensive, systemic-level characterization in the Central Asian region of the intestinal barrier status, local inflammation levels, and the fine structure of the microbiome in children with autism spectrum disorder (ASD). It demonstrates that these factors are deeply involved in the pathogenesis and formation of the clinical phenotype of the disorder. The pathophysiological basis for gastrointestinal disturbances in ASD involves a combined process of increased intestinal mucosal permeability (indicated by elevated zonulin levels) and chronic inflammation (indicated by high calprotectin levels).