Systematic structure-based analysis of RET variants in MEN2A and Hirschsprung's disease, and the paradoxical co-occurrence of both conditions.

Variants in the human receptor tyrosine kinase RET can cause RET loss-of-function and Hirschsprung's disease (HSCR), while activating RET variants drive cancers including multiple endocrine neoplasia type 2 (MEN2). Paradoxically, some variants cause both HSCR and MEN2A. We curated 77 RET extracellular positions associated with HSCR, MEN2A or both and used a structure-based approach to predict the effects of variants at these positions on RET structure. Approximately 90% of HSCR-associated positions can, upon mutation, disrupt intramolecular interactions stabilizing RET tertiary structure via distinct mechanisms. Only a minority perturb protein-protein interactions needed for signal activation. In contrast, our analysis showed that ∼75% of variants causing MEN2A lead to an unpaired cysteine that can form an intermolecular disulfide bond between two RET monomers. Other MEN2A variants are likely to enhance RET homodimerization via membrane-proximal extracellular interactions. Substitutions that, concurrently, destabilize RET structure and result in an unpaired cysteine are predicted to cause the paradoxical co-occurrence of HSCR and MEN2A. Our findings lay out a mechanistic basis for almost all identified pathological RET mutations, and suggest therapeutic strategies for targeting RET activity in HSCR and MEN2A.