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Analysis of a novel AVPR2 mutation in a family with nephrogenic diabetes insipidus.

ABSTRACT: Congenital nephrogenic diabetes insipidus (NDI) is a rare X-linked recessive disorder associated with germ-line mutations of the arginine vasopressin (AVP) receptor type 2 (AVPR2) gene. Recent molecular studies have demonstrated that insensitivity of renal tubule cells to AVP is associated with AVPR2 mutations. We identified a novel deletion mutation at nucleotide position 302 (302delC), in a Korean NDI family, that results in a frameshift and a truncated receptor protein. To identify the mutant AVPR2 protein we developed an expression vector for the AVPR2 mutation by a PCR-based restriction fragment replacement strategy. COS-7 cells were transiently transfected with expression vectors for the wild-type and mutant genes, and we analyzed AVP-induced cyclic adenosine monophos-phate (cAMP) responses, and assessed the localization of AVPR2 receptors, in the transfected COS-7 cells. In the cells expressing the mutant gene, the maximum AVP-induced cAMP response was reduced and the truncated receptor proteins were retained within the cytoplasmic compartment. These results suggest that the novel frameshift AVPR2 (302delC) mutation is responsible for the AVP resistance in the family with congenital NDI.

SUBMITTER: Moon SD

PROVIDER: S-EPMC3048978 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Analysis of a novel AVPR2 mutation in a family with nephrogenic diabetes insipidus.

Moon Sung-Dae SD Kim Ju-Hee JH Shim Joo-Yun JY Lim Dong-Jun DJ Cha Bong-Yun BY Han Je-Ho JH

International journal of clinical and experimental medicine 20101130 1

Congenital nephrogenic diabetes insipidus (NDI) is a rare X-linked recessive disorder associated with germ-line mutations of the arginine vasopressin (AVP) receptor type 2 (AVPR2) gene. Recent molecular studies have demonstrated that insensitivity of renal tubule cells to AVP is associated with AVPR2 mutations. We identified a novel deletion mutation at nucleotide position 302 (302delC), in a Korean NDI family, that results in a frameshift and a truncated receptor protein. To identify the mutant ...[more]

PMID: 21394280

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Project description:Background: Congenital nephrogenic diabetes insipidus (CNDI) is a rare inherited disease that is caused by mutations in arginine vasopressin receptor 2 (AVPR2) or aquaporin 2 (AQP2). Functional analysis of the mutated receptor is necessary to verify the impact of the mutation on receptor function and suggest some possible therapeutic strategies for specific functional defects. Methods: Family history and clinical information were collected. Whole-exome sequencing and sanger sequencing were performed to determine the potential genetic cause of diabetes insipidus. The identified variant was classified according to the American College of Medical Genetics (ACMG) criteria. Bioinformatic analysis was performed to predict the function of the identified variation. Moreover, wild-type and mutated AVPR2 vectors were constructed and transfection to HEK-293T cells. Immunofluorescence experiments were performed to investigate the expression and localization of the mutated protein and cAMP parameter assays were used to measure its activity in response to AVP. Results: The heights of the adult members affected with polyuria and polydipsia were normal, but all affected children had growth retardation. Next-generation sequencing identified a novel mutation in AVPR2 gene (c.530T > A) in this family. Bioinformatic analysis indicated that the mutation in AVPR2 changed the hydropathic characteristic of the protein and was probably deleterious. Although immunofluorescence showed that the mutated AVPR2 was normally expressed in the cell surface, the intracellular cAMP concentration stimulated by AVP was significantly lower in cells transfected with mutated AVPR2 than cells transfected with wild-type AVPR2. Based on the ACMG criteria, the novel c.530T > A variant of the AVPR2 gene was likely pathogenic and the affected family members were diagnosed as CNDI. After the confirmation of the diagnosis, the proband was treated with compound amiloride hydrochloride and rhGH, the symptoms of polyuria, polydipsia and growth retardation were all improved. Conclusion: These findings suggested that the novel mutation in AVPR2 (c.530T > A) was a true disease-causing variant with mild effects, which could be classified as a type III mutant receptor. Moreover, investigations of the function of growth hormone axis could be important for the pediatric CNDI patients with extreme short stature, and rhGH treatment might improve the final adult heights in these patients.

Project description:BackgroundCongenital nephrogenic diabetes insipidus (NDI) is characterised by an inability to concentrate urine despite normal or elevated plasma levels of the antidiuretic hormone arginine vasopressin. We report a Japanese extended family with NDI caused by an 11.2-kb deletion that includes the entire AVPR2 locus and approximately half of the Rho GTPase-activating protein 4 (ARHGAP4) locus. ARHGAP4 belongs to the RhoGAP family, Rho GTPases are critical regulators of many cellular activities, such as motility and proliferation which enhances intrinsic GTPase activity.ARHGAP4 is expressed at high levels in hematopoietic cells, and it has been reported that an NDI patient lacking AVPR2 and all of ARHGAP4 showed immunodeficiency characterised by a marked reduction in the number of circulating CD3+ cells and almost complete absence of CD8+ cells.MethodsPCR and sequencing were performed to identify the deleted region in the Japanese NDI patients. Immunological profiles of the NDI patients were analysed by flow cytometry. We also investigated the gene expression profiles of peripheral blood mononuclear cells (PBMC) from NDI patients and healthy controls in microarray technique.ResultsWe evaluated subjects (one child and two adults) with 11.2-kb deletion that includes the entire AVPR2 locus and approximately half of the ARHGAP4. Hematologic tests showed a reduction of CD4+ cells in one adult patient, a reduction in CD8+ cells in the paediatric patient, and a slight reduction in the serum IgG levels in the adult patients, but none of them showed susceptibility to infection. Gene expression profiling of PBMC lacking ARHGAP4 revealed that expression of RhoGAP family genes was not influenced greatly by the lack of ARHGAP4.ConclusionThese results suggest that loss of ARHGAP4 expression is not compensated for by other family members. ARHGAP4 may play some role in lymphocyte differentiation but partial loss of ARHGAP4 does not result in clinical immunodeficiency.

Dataset Information

Analysis of a novel AVPR2 mutation in a family with nephrogenic diabetes insipidus.

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Analysis of a novel AVPR2 mutation in a family with nephrogenic diabetes insipidus.

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