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

Background The endocannabinoid system (ECS) regulates key processes in energy homeostasis, but its contribution to severe obesity beginning in childhood or adolescence remains incompletely understood. This study aimed to identify rare variants in ECS genes and evaluate their potential structural effects. Methods A total of 93 adults with severe obesity beginning before 21 years of age (BMI ≥ 35 kg/m²) were recruited from two specialized obesity programs; among participants with onset information, 97.8% reported childhood onset. Whole-exome sequencing was performed, followed by targeted analysis of ECS-related genes. Rare variants (allele frequency <1%) were confirmed by Sanger sequencing and assessed using in silico pathogenicity prediction and molecular modeling. In silico saturation mutagenesis of all 19 possible substitutions at each variant position was additionally performed using three complementary stability predictors based on different methodological principles (FoldX, Pythia and DDMut), together with AlphaMissense. Results Nine rare variants were identified in twelve individuals, seven of which were defined as variants of uncertain significance (VUS). Among the observed variants, FAAH p.(Arg326Pro) showed the most concordant predicted impact: it ranked as the most destabilizing substitution at position 326 by FoldX (ΔΔG = +3.04 kcal/mol; rank 1 of 19) and was the only observed variant classified as likely pathogenic by AlphaMissense (0.884). Arg326 lies 24.3 Å from the catalytic triad (Lys142–Ser217–Ser241), consistent with a predicted effect on fold stability rather than direct disruption of the active site. Conversely, at DAGLA position 750, all 19 substitutions were predicted as neutral by FoldX and DDMut and as likely benign by AlphaMissense, consistent with predicted structural tolerance at this position. No anthropometric or metabolic parameter differed significantly between carriers and non-carriers after correction for multiple testing. Conclusions Rare ECS variants, including a previously unreported FAAH p.(Arg326Pro) substitution, were identified in adults with severe obesity, predominantly with childhood onset. These in silico analyses are predictive and provide computational evidence for prioritizing candidate variants, notably FAAH p.(Arg326Pro); they do not, by themselves, establish pathogenicity or a causal relationship with the phenotype, which will require functional validation.