Rare endocannabinoid system variants identified by whole-exome sequencing and structural modeling in adults with severe obesity predominantly of childhood onset.

Salum K.; de Souza R.; Antunes D.; Assis I.; Gouvêa L.; Teixeira M.; de Melo M.; Palhinha L.; Abreu G.; Alves N. et al. · Frontiers in endocrinology · 2026

**Background & Methods** This whole-exome sequencing study recruited 93 adults with severe obesity (BMI ≥35 kg/m²) with onset before age 21 years (97.8% with childhood onset) from specialized obesity programs. Rare variants (allele frequency <1%) in endocannabinoid system (ECS) genes were identified and evaluated using Sanger sequencing confirmation, in silico pathogenicity prediction, molecular modeling, and saturation mutagenesis analysis with multiple stability predictors (FoldX, Pythia, DDMut, and AlphaMissense). **Key Findings** • Nine rare variants were identified in 12 individuals across ECS-related genes, with seven classified as variants of uncertain significance (VUS); notably, a previously unreported FAAH p.(Arg326Pro) substitution showed the most concordant predicted pathogenic impact across multiple prediction tools. • FAAH p.(Arg326Pro) ranked as the most destabilizing substitution at position 326 by FoldX analysis (ΔΔG = +3.04 kcal/mol; rank 1 of 19 possible substitutions) and was uniquely classified as likely pathogenic by AlphaMissense (score 0.884), with predicted effects on protein fold stability rather than direct active site disruption. • In contrast, DAGLA position 750 variants demonstrated structural tolerance, with all 19 possible substitutions predicted as neutral by FoldX and DDMut and likely benign by AlphaMissense, suggesting limited pathogenic potential at this locus. **Dosage & Administration** Not reported. **Safety & Adverse Effects** Not reported. **Evidence Quality** This study represents early-stage genetic discovery research with significant limitations. The lack of significant differences in anthropometric or metabolic parameters between variant carriers and non-carriers after multiple testing correction undermines evidence for phenotypic association. The in silico predictive analyses provide computational support for variant prioritization but do not establish pathogenicity or causality. The authors acknowledge that functional validation studies are necessary to confirm pathogenic mechanisms. The modest sample size (93 participants, only 12 with identified variants) limits generalizability. As a review article presenting novel genetic findings, this work represents hypothesis-generating evidence requiring independent replication and functional studies before clinical application. No direct therapeutic interventions or clinical outcomes were evaluated.

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