2004 Global Researcher Conference Proceeding

April 09 - 11, 2004

Conference: 2004 Global Researcher Conference
Title: Clinical Phenotype and Molecular Characterization of A Mutant V2 Receptor Associated with Partial Congenital Nephrogenic Diabetes Insipidus
Authors: Rittig, Soren; Faerch, M; Christensen, JH; Kamperis, K.; Corydon, TH; Gregersen, N.; de Zegher, F.; Proesmans, W.
Institutions: Skejby University Hospital, Aarhus University Hospital, University Hospital Leuven, University of Aarhus, University of Leuven

A large number of mutations in the renal vasopressin V2 receptor are known to cause congenital nephrogenic diabetes insipidus (CNDI) with an X-linked recessive inheritance. Neither the pathogenic mechanism of the disease nor the full structure-function relationship of the receptor is fully understood. One possible way to increase our understanding of such is to study mutations that are associated with unusual mild phenotypes (partial CNDI). We studied clinically and genetically a Belgian kindred with partial CNDI where two affected boys initially were interpreted as having neurohypophyseal DI as intranasal dDAVP in normal doses allegedly alleviated their polyuria and polydipsia. We performed clinical and genetic studies on the two affected boys, their mother, and a healthy sibling without polyuric symptoms. Furthermore, we expressed the identified mutation in human embryonic kidney cells to study the in vitro properties of the mutation.

The AVP-NPII gene was normal in all 4 subjects. However, we identified a missense mutation in the AVPR2 gene, i.e. C1524A leading to substitution of Ser329 with Arg in both affected brothers and their mother (heterozygous). The un-affected sibling had normal AVPR2 gene.

The affected boys both had a mild DI phenotype with moderate polyuria (96 and 120 ml/kg BW/24h) with reduced urinary osmolality (253 and 222 mosm/kg). Their basal p-AVP levels were about 10-fold elevated consistent with CNDI. During fluid deprivation they were both able to concentrate urine to almost normal levels (max. 573 and 720 mosm/kg). However, they both had a subnormal response to infusion of high doses of AVP and dDAVP, with a shift to the right in the relation of urine osmolality to plasma VP. The heterozygous mother and the unaffected sibling showed normal urinary concentrating ability.

Western blotting showed no significant difference in protein production suggesting that similar amounts of receptor protein are produced in both mutant and WT cells. Immunostaining and confocal laser scanning microscopy established that WT receptors were localized mainly on the cell surface whereas the majority of the mutant receptors seemed to accumulate in a diffuse pattern within the cells partly co-localizing with the endoplasmic reticulum. Ligand binding assay with radiolabeled AVP demonstrated no significant difference in AVP affinity (1/Kd) between the WT and mutant receptors. However, the Bmax of the mutant receptor was significantly lower further indicating that the surface expression is reduced compared to WT. Displacement analysis with AVP or dDAVP demonstrated a slightly lower affinity of both WT and mutant receptors towards dDAVP than AVP. Conclusion: The S329R substitution of the V2 receptor is associated with a very partial CNDI phenotype and seems not to affect the affinity of the V2 receptor towards AVP and dDAVP but rather the efficiency of its intracellular trafficking.

Rittig, et al., studied a family wherein two of the sons exhibited mild symptoms of NDI and one son was symptom free. They found that the mother and the two sons with congenital NDI all carried the same mutation in the vasopressin-2 receptor (AVPR2) gene. This mutation resulted in AVPR2 proteins with an arginine amino acid where a serine amino acid should have been. This resulted in the vasopressin 2 receptor (AVP2R) not being able to transfer itself normally from the cell interior to the cell membrane. Thus, even though the mutant V2Rs could bind with arginine vasopressin (AVP) or its synthetic counterpart, dDAVP in laboratory cell cultures, in the cells of the affected family, they could not to a sufficient degree reach the cell membrane, where they must be if they are to bind with AVP. This type of mutation is unique in that it causes very mild symptoms.