RESUMEN
BACKGROUND: X-linked nephrogenic diabetes insipidus (NDI) is a rare polyuric disorder caused by inactivating mutations in the arginine vasopressin receptor Type 2 (AVPR2) gene. METHODS: NDI patients from six unrelated families were subjected to mutational analysis of the AVPR2 gene. In-depth in vitro characterization of novel AVPR2 mutants by a combination of functional and immunological techniques provided further insight into molecular mechanisms causing receptor dysfunction. RESULTS: Mutational analysis revealed four novel (A89P, G107R, Q174R, W208X) and three recurrent (V277A, R337X, ΔR247-G250) mutations within the AVPR2 gene. One family carried the missense mutation R337X and a 12-bp deletion (ΔR247-G250), corresponding to a fragment in the third intracellular loop (ICL3), which was not genetically linked to R337X. The functionally tested missense mutations A89P, G107R and Q174R led to reduced receptor cell surface expression in transfected COS-7 cells, most probably due to misfolding and intracellular retention, and consequently to reduction or loss of agonist-mediated cyclic adenosine monophosphate formation. Deletion of R247-G250 had no effect on receptor function in vitro. Comparison with other mammalian AVPR2 orthologs showed that this part of the ICL3 is structurally not conserved and, therefore, less relevant for receptor function. In contrast, all missense mutations (A89P, G107R, Q174R, V277A) affect receptor positions that were fully preserved during mammalian evolution. CONCLUSION: Our results provide valuable information about residues critical for AVPR2 folding, trafficking and function and proof that these mutations are responsible for causing NDI in the affected subjects.