Clinical, Genetic and Functional Characterization of a Novel AVPR2 Missense Mutation in a Woman with X-Linked Recessive Nephrogenic Diabetes Insipidus

Abstract

Nephrogenic diabetes insipidus (NDI) is a rare disorder characterized by renal unresponsiveness to the hormone vasopressin, leading to excretion of large volumes of diluted urine. Mutations in the arginine vasopressin receptor-2 (AVPR2) gene cause congenital NDI and have an X-linked recessive inheritance. The disorder affects almost exclusively male family members, but female carriers occasionally present partial phenotypes due to skewed inactivation of the X-chromosome. Here, we report a rare case of a woman affected with X-linked recessive NDI, presenting an average urinary output of 12 L/day. Clinical and biochemical studies showed incomplete responses to water deprivation and vasopressin stimulation tests. Genetic analyses revealed a novel heterozygous missense mutation (c.493G > C, p.Ala165Pro) in the AVPR2 gene. Using a combination of in-silico protein modeling with human cellular models and molecular phenotyping, we provide functional evidence for phenotypic effects. The mutation destabilizes the helical structure of the AVPR2 transmembrane domains and disrupts its plasma membrane localization and downstream intracellular signaling pathways upon activation with its agonist vasopressin. These defects lead to deficient aquaporin 2 (AQP2) membrane translocation, explaining the inability to concentrate urine in this patient.

Keywords: AVPR2; case report; genetics; nephrogenic diabetes insipidus; vasopressin.

Figure 1

Biochemical and genetic studies of the patient with nephrogenic diabetes insipidus (NDI). (A) Pedigree of the patient with NDI. The inheritance follows a typical X-linked recessive pattern, apart from the reported female patient (red arrow). Individuals are represented as males (squares), females (circles), affected (filled symbol), unaffected (open symbol) and deceased (oblique line through symbol). (B,C) Water-deprivation and dDAVP stimulation tests. The water-deprivation test was continued until the patient lost 5% of her body weight, which occurred after 6 h (B). At the end of the test, serum osmolality had increased from 276 to 311 mOsm/Kg (normal 275–295) and the urine osmolality had increased from 95 to only 191 mOsm/Kg (normal 300–900) (C). Arrows indicate the administration of dDAVP (2 μg intramuscular injection), which did not increase urine osmolality, thereby supporting the diagnosis of NDI. (D) Extra-renal responses to dDAVP. Arrow indicates the administration of dDAVP (0.3 μg/kg, intravenous infusion over 20 min). Mean arterial blood pressure decreased 18% (normal > 10%), heart rate increased 10% (normal > 20%), plasma renin activity increased 4% (normal > 65%), factor VIIIc increased 2.2× (normal > 3×), von Willebrand factor increased 1.5× (normal > 2×). (E) Response to treatment. Treatment with increasing doses of dDAVP did not significantly improve the polyuria. Treatment with hydrochlorothiazide (HCTZ) 50 mg bid, amiloride chloridrate (Amil) 5 mg bid and a low-sodium diet, resulted in a reduction of diuresis to approximately 5 L/day. (F) Genomic structure (Xq28) showing the exons (grey boxes), introns (white boxes), 3′- and 5′- untranslated regions (UTR) (black boxes) of the AVPR gene. The locations of the PCR amplified fragments and the identified mutation are shown. (G) Partial DNA sequencing of exon 2 of the AVPR2 gene in the patient and a control. We identified a novel heterozygous missense mutation (c.493G > C, p.Ala165Pro; noted by an asterisk) in the patient. (H) Agarose gel electrophoresis of MwoI-digested PCR fragments. The mutation creates a restriction site for this enzyme, resulting in fragments of 200 base pairs (bp) and 83 bp (not shown), whereas the normal allele results in fragments of 283 bp. The patient is heterozygous for the mutation, the father is hemizygous and the mother is homozygous for the normal allele. We used a 100 bp ladder as a DNA size marker.

Figure 2

Model structure with key regions of human AVPR2 and Ala165Pro-AVPR2. (A) Lateral view of the 3D protein model of the wild-type human AVPR2, including seven transmembrane domains/helices (H1–7) connected with three intracellular (I1–3) and extracellular (E1–3) loops. Dark blue sphere: Alanine residue at position 165 (A165). (B) Surface mapping of the vasopressin (AVP) ligand-binding pocket in AVPR2. (C) Key residues in the AVP binding region and conserved residues of rhodopsin-like GPCR family. (D) Lateral view of the 3D protein model of the mutant Ala165Pro-AVPR2. (E) Molecular interaction networks around residue 165 in the WT-AVPR (left panel) and the mutant Ala165Pro-AVPR2 (right panel). Dark blue sphere: Alanine (A) residue at position 165 (A165); Purple blue sphere: Proline (P) residue at position 165 (P165); Cyan sticks and surface: AVP binding pocket; Orange sticks: conserved residues; Magenta sticks: -binding residues; Grey sticks: other residues in the network; Black dash: hydrogen bonds. Residues are indicated by their position number, preceded by the corresponding amino-acid letter: A, Alanine; F, Phenylalanine; H, Histidine; L, Leucine; M, Methionine; N, Asparagine; P, Proline; S, Serine; V, Valine; W, Tryptophan.

Figure 3

A165P mutant impairs the cell surface localization of AVPR2. (A) HEK293 cells were transfected with HA-tagged WT-AVPR2 (WT) or Ala165Pro-AVPR2 (Mut). At 36 h after transfection, cells were harvested, lysed and proteins were analyzed by immunoblotting (left panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Semiquantitative analysis of the effects of the Ala165Pro mutation on the total levels of AVPR2 protein was performed by densitometry (right panel). (B) Crude plasma membrane extracts were prepared from cells transfected with HA-tagged WT or Mut AVPR2 and proteins were analyzed by immunoblotting (left panel). β-actin was used as a loading control. A semiquantitative analysis of the effects of Ala165Pro mutation on the plasma membrane levels of AVPR2 protein was performed by densitometry (right panel). (C) In parallel, to analyze the cell surface expression of AVPR2, HA-tagged WT or Mut AVPR2 expressing cells were biotinylated using Sulfo-NHS-SS-Biotin and then precipitated with streptavidin-Sepharose beads. Immunoblotting was performed with respective antibodies to visualize the surface expression of proteins (left panel). Plasma membrane Ca²⁺ ATPase (PMCA) was used as a loading control. A semiquantitative analysis of the effects of Ala165Pro mutation on the cell surface abundance levels of AVPR2 protein was performed by densitometry (right panel). (D) HEK293 cells were transfected with GFP-tagged WT or Mut AVPR2. At 36 h after transfection, cells were incubated with α-GFP antibody for 1 h at 4 °C followed by cell fixation and incubation with Alexa 547 conjugated secondary antibody. The cellular localization of AVPR2 was visualized through immunofluorescence microscopy (40× magnification). Consistent with the immunoblotting results above, the Mut displays less membrane staining when compared to the WT. Green, GFP staining indicates the intracellular and membrane location; red, α-GFP staining indicates only the membrane location; yellow, co-localization between the green GFP and red α-GFP staining. Values in all bar charts are given in means ± SEM (n = 3). Asterisks indicate significant differences (unpaired t-tests with Welch’s correction, two-tail): * p ≤ 0.05; ns, not significant.

Figure 4

Ala165Pro-AVPR2 mutant impairs the activation of AVP-dependent downstream signaling pathways. (A,B) HEK293 cells were transiently transfected with WT-AVPR2 (WT) or Ala165Pro-AVPR2 (Mut). After 36 h, the cells were stimulated with dimethyl sulfoxide (DMSO) control (−dDAVP) or 1 μM dDAVP (+dDAVP) for 10 min and processed for quantification of intracellular cAMP (A) and PKA kinase activity. (B) The Mut AVPR2 displays lower cAMP and PKA activation levels than the WT. (C,D) WT and Mut AVPR2 expressing cells were stimulated with DMSO control (−dDAVP) or 250 nM dDAVP (+dDAVP) for 60 min. Cells were then harvested, lysed and proteins were analyzed by immunoblotting using antibodies against total and phosphorylated (p-) PKA (C) and p38-MAPK (D) (left panels). Phosphorylated protein abundance was normalized against the respective total proteins and a semiquantitative analysis of the effects of Ala165Pro phosphorylation was done by densitometry (right panels). (E) WT and Mut AVPR2 expressing cells were stimulated with DMSO control (−dDAVP) or 250 nM dDAVP (+dDAVP) for 60 min. Following the treatment, cells were biotinylated using Sulfo-NHS-SS-Biotin and then precipitated with streptavidin-Sepharose beads. Immunoblotting was performed with antibodies against AQP2 to visualize its surface expression (left panel). PMCA was used as a loading control. A semiquantitative analysis of the effects of Ala165Pro mutation on the plasma membrane levels of AVPR2 protein was done by densitometry (right panel). Values in all bar charts are given in means ± SEM (n = 3). Asterisks indicate significant differences (one-way ANOVA; Tukey multiple comparisons correction): * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.001; ns, not significant.