Functional analyses of mutations found in AVP-NPII gene of patients diagnosed with diabetes insipidus

In healthy individuals, blood osmolality is kept within certain limits through mechanisms that regulate body water balance. Thirst is stimulated by sending signals to the kidneys for water protection with an increase in plasma osmolality or a decrease in blood volume. As a result of this stimulation, the arginine vasopressin hormone (AVP, ADH, antidiuretic hormone) which is stored in the posterior pituitary, is released into the blood. The AVP hormone reaches to the kidneys through the bloodstream and binds to arginine vasopressin receptor 2 (AVPR2) in the collection duct cells. The binding of AVP to the AVPR2 leads to the aquaporin 2 water channel proteins (AQP2) to be phosphorylated and settle in the apical membrane. The reabsorption of the required water takes place through the AQP2 water channel, thus the water balance in the body is reestablished. Any malfunction that may occur within this mechanism causes disruption of the body's water balance and the development of Diabetes insipidus (DI) disease, which is characterized by polyuria and polydipsia. DI can be either central type (CDI) due to inadequate synthesis and/or release of AVP hormone or nephrogenic type (NDI) due to inadequate response of the kidney to AVP. CDI can be either acquired by damage to the neurohypophysis region as a result of head injuries, brain tumors, surgical operations or various diseases or can be congenital due to mutations in the vasopressin-neurophysin II (AVP-NPII) gene. In many studies concerning AVP-NPII, it has been found that the precursor hormone fails during proper folding and processing due to mutations in the AVP-NPII gene. As a consequence, the mutant protein accumulates in the endoplasmic reticulum (ER), generates protein aggregations and leads to the death of magnocellular nerve cells by preventing the regular processing of other essential proteins. The aim of this thesis is to perform functional analyzes of G45C, 207_209delGGC and G88V mutations defined in the AVP-NPII gene of patients diagnosed with DI, which are not included in the literature. In the experimental studies, the relevant mutations on the expression vector containing the wild-type AVP-NPII gene were created using the site-directed mutagenesis method and transient transfection of these vectors to Neuro2A cells was performed. The amount of wild type and mutant AVP that were released into the medium by Neuro2A cells were determined by two separate methods, namely radioimmunoassay (RIA) and Enzyme-Linked ImmunoSorbent Assay (ELISA). Fluorescence imaging studies were performed to determine the differences in the intracellular traffic of wild-type and mutant precursor hormones. In addition, since the RIA method used in determining the amount of AVP is a difficult and long-lasting method, the usability of the ELISA method, which provides faster results and is easy to perform as an alternative method for RIA, was evaluated by the Bland-Altman method, which is a statistical analysis. As a result of the study, it was determined that G45C and G88V mutant proteins were significantly reduced in the cell culture medium compared to wild type AVP-NPII protein and were trapped in the ER in the cell. However, it was observed that the amount of 207_209delGGC mutant protein is similar to the wild type protein in the cell culture medium and was distributed in intracellular compartments by not being trapped in ER, likewise the wild type. It has been concluded that G45C and G88V mutations have significant effects in processes such as the proper folding, gain of three-dimensional structure, processing, or intracellular traffic of the precursor hormone, and the 207_209delGGC mutation does not have any significant effect in these processes. In addition, it was determined that ELISA method can be used as an alternative method instead of RIA method in determining the amount of AVP.