Structural distance at the tRNA synthetase active site interface predicts pathogenicity but is captured by AlphaMissense and EVE except among score-ambiguous variants

Variants of uncertain significance have accumulated as genomic sequencing has become more widespread, which complicates rare disease diagnosis and requires substantial resources for re-evaluation. Aminoacyl-tRNA synthetases (ARSs) are a protein family with extensive variant data and well-characterized disease associations, making them an ideal system for investigating the relationship between variant location and pathogenicity. Using structural distance measurements to the ARS-tRNA binding interface combined with existing pathogenicity predictors, AlphaMissense and EVE, we investigated whether explicit structural binding information could improve missense variant pathogenicity prediction. Pathogenic variants were found to cluster significantly closer to the tRNA-binding interface than benign variants (p = 0.0003). Incorporating explicit distance information into a Bayesian mixture model did not substantially improve predictive performance over AlphaMissense and EVE alone, suggesting that these models already implicitly capture relevant structural binding context. However, a clinically important subset of interface variants classified as ambiguous by both existing models identifies a specific gap where explicit structural distance information may provide added discriminative value, but the limited number of clinically validated variants currently available constrains the ability to fully evaluate this potential. Incorporating additional biologically relevant features not captured by existing models, such as protein stability or conformational dynamics, as well as refining structural distance calculations, may further improve classification of this subset. These findings highlight both the power and the limitations of existing pathogenicity predictors and suggest that structurally informed approaches targeting the binding interface represent a promising direction for improving classification of these ambiguous variants that have great clinical significance. Author SummaryAdvances in clinical genetic sequencing have caused increasing identification of genetic variants whose impact on human health is unknown. These "variants of uncertain significance" present a major challenge because their role in causing disease cannot yet be confirmed or ruled out. This study focuses on a specific family of essential enzymes called aminoacyl-tRNA synthetases, which play a critical role in the process of proteins translation. Mutations in these enzymes have been linked to a range of diseases. This project aims to provide a novel method for determining pathogenicity of variants specifically in aminoacyl-tRNA synthetases. We propose that physical proximity of a variant to the functional binding site of the enzyme is influential in determining pathogenicity. We find that this spatial relationship is a meaningful indicator of a variants potential to disrupt normal function.