Functional characterization of a large deletion in AVPR2 gene causing severe nephrogenic diabetes insipidus in a Turkish patient

SAĞLAR ÖZER E., Erdem B., Deniz F., MERGEN H.

INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol.9, no.8, pp.16199-16205, 2016 (SCI-Expanded) identifier identifier


Objective: The main purpose of this study is to characterize functionally a large deletion in arginine vazopression type-2 receptor (AVPR2) gene. Changes in AVPR2 gene mostly lead to a rare hereditary polyuric disease, X-linked nephrogenic diabetes insipidus (NDI). The disease is characterized by the extreme production of urine and the patients have problems with concentrating the urine in response to the antidiuretic hormone vasopressin. In our previous study we have identified a novel 388 bp deletion in the AVPR2 gene in a patient with NDI and in his family. Methods: For functional analyze studies, identified deletion was re-created by PCR based site-directed mutagenesis and restriction fragment replacement strategy based on DNA sequence and expressed in COS7 cells. We performed total and surface ELISA assay and cAMP assay for assessing the ability of transfected cells to produce cAMP in response to stimulation with dDAVP. Fluorescence staining was performed for determining the cell trafficking of the mutant protein. Results: Results of functional characterization of 388 bp deletion have revealed that mutant V2R did not show any expression on the cell surface compared to the wild type receptor while showed reduced cellular expression in total (31.93%+/- 8.8) compared to the wild type receptor. cAMP accumulation assay results are supported the ELISA results of the mutant receptor protein. Conclusions: In conclusion, our results will provide valuable information about the AVPR2 trafficking and function of the mutant protein and we believe that our study will contribute to shedding light on mechanisms of molecular pathology of AVPR2 deletions.