A Novel Polymorphism in the Coding Region of the Vasopressin Type 2 Receptor Gene

Title: A Novel Polymorphism in the Coding Region of the Vasopressin Type 2 Receptor Gene
Authors: Rocha, Juliane L.; Moreira, A.C.; Friedman, Eitan; Liberman, Bernardo; Silva, B.C.; De Marco, Luiz
Publisher: Brazilian Journal of Medical and Biological Research
Date Published: April 01, 1997
Reference Number: 181
Nephrogenic diabetes insipidus (NDI) is a rare disease characterized by renal inability to respond properly to arginine vasopressin due to mutations in the vasopressin type 2 receptor (V2(R)) gene in affected kindreds. In most kindreds thus far reported, the mode of inheritance follows an X chromosome-linked recessive pattern although autosomal-dominant and autosomal-recessive modes of inheritance have also been described. Studies demonstrating mutations in the V2(R) gene in affected kindreds that modify the receptor structure, resulting in a dys- or nonfunctional receptor have been described, but phenotypically indistinguishable NDI patients with a structurally normal V2(R) gene have also been reported. In the present study, we analyzed exon 3 of the V2(R) gene in 20 unrelated individuals by direct sequencing. A C--62 T alteration in the third position of codon 331 (AGC--> AGT), which did not alter the encoded amino acid, was found in nine individuals, including two unrelated patients with NDI. Taken together, these observations emphasize the molecular heterogeneity of a phenotypically homogeneous syndrome.

The following article is presented with the permission of the publisher for educational use within the NDI community. No part of this article may be reproduced in any way without permission in writing from the publisher.

Germline mutations in the vasopressin type 2 receptor (V2(R)) gene have been demonstrated to co-segregate with nephrogenic diabetes insipidus, a rare disorder characterized by the inability to concentrate the urine due to a renal insensitivity to vasopressin.

To our knowledge, only two neutral polymorphisms have been demonstrated in this receptor gene (1,2). In addition, an A61V mutation that does not alter vasopressin binding or the stimulation of adenylate cyclase activity was also demonstrated (3). Here, we report the presence of a new polymorphism in 7 healthy Brazilian individuals, as well as in two unrelated patients with nephrogenic diabetes insipidus.

High molecular DNA was isolated from peripheral blood leukocytes according to a standard protocol (4). Based on the genomic sequence of the V2(R) gene(5), sense (5'-CAT CCT GAA CCC AAC CTA C-3') and antisense (5'-CTC TAG AGG CAA GAC ACC C-3') primers were used, as previously described (6). The polymerase chain reaction (PCR) was used to amplify this 270-bp fragment, encompassing the second intronic region and the 3' untranslated region. PCR conditions were as previously described (6). The amplified fragments were visualized by silver staining after electrophoresis on 5% polyacrylamide gel to ascertain the correct size. For direct sequencing of PCR products, the Sequenase PCR Product Sequencing kit (United States Biochemical, Cleveland, OH) was used, according to manufacturer instructions.

Figure 1  Direct sequence analysis of the amplified genomic fragment of exon 3 of the V2(R) gene showing a polymorphism* (AGT; right panel) as well as the published sequence (AGC; left panel) of codon 331.

The data in Figure 1 show the presence of a C–>T alteration in the third base of codon 331 for serine (AGC-->AGT). This homozygous alteration was found in 25% (5/20) of all individuals, including two of the three individuals with nephrogenic diabetes insipidus, thus representing a polymorphism. Four individuals were heterozygous (AGC/AGT). This alteration did not introduce or abolish any restriction site.

To date, 67 mutations have been described in unrelated nephrogenic diabetes insipidus families throughout the world (7,8). However, no significant correlation of phenotype expression with genotypic variation has been demonstrated. The point mutation (A61V) that resulted in the substitution of one hydrophobic amino acid within the transmembrane region (3) was a rare polymorphism and did not cause the disease in that patient. Therefore, possible structural variations in the sequence of a normal V2(R) gene are not accounted for in all cases of nephrogenic diabetes insipidus. Indeed, another gene, the vasopressin-regulated water channel aquaporin-2 gene, has been associated with the development of autosomal nephrogenic diabetes insipidus (9,10). The identification of a second gene involved in the development of nephrogenic diabetes insipidus clearly indicates that linkage analysis should be performed before initiating mutation analysis to define which gene should be analyzed. Despite the similar clinical phenotype of all cases of nephrogenic diabetes insipidus, the analysis of several mutations reported here and the previously described polymorphisms has not shown the presence of a hot spot for these alterations, although most mutations are located in the transmembrane domain of the gene. Detection of polymorphic regions in the genes involved in the development of the disease may provide information about mutable areas within these genes.

In summary, we described here the third polymorphism in the vasopressin type 2 receptor gene, indicating that the possible structural variations in the sequence of this gene could be used, together with linkage analysis, to help understand the pathophysiology of the hereditary forms of nephrogenic diabetes insipidus.

  1. Pan Y, Metzenberg A, Das S, Jing B & Gitschier J (1992). Mutations in the V2 vasopressin receptor gene are associated with X-linked nephrogenic diabetes insipidus. Nature Genetics, 2: 103-106.
  2. Friedman E, Carson E, Larsson C & De Marco L (1993). A polymorphism in the coding region of the vasopressin type 2 (V2(R)) gene. Human Molecular Genetics, 2: 1746.
  3. Pan Y, Wilson P & Gitschier J (1994). The effect of eight V2 vasopressin receptor mutations on stimulation of adenylyl cyclase and binding to vasopressin. Journal of Biological Chemistry, 269: 31933-31937.
  4. Jeanpierre M (1987). A rapid method for the purification of DNA from blood. Nucleic Acids Research, 15: 9611.
  5. Birnbaumer M, Seibold A, Gilbert S, Ishido M, Barberis C, Antaramian A, Brabet P & Rosenthal W (1992). Molecular cloning of the receptor for human antidiuretic hormone. Nature, 357: 333-335.
  6. Friedman E, Bale AE, Carson E, Boson W, Nordenskjöld M, Ritzén M, Ferreira PC, Jammal AR & De Marco L (1994). Nephrogenic diabetes insipidus: an X chromosome-linked dominant inheritance pattern with a vasopressin type 2 receptor gene that is structurally normal. Proceedings of the National Academy of Sciences, USA, 91: 8457-8461.
  7. Bichet DG, Birnbaumer M, Lonergan M, Arthus M-F, Rosenthal W, Goodyear P, Nivet H, Benoit S, Giampietro P, Simonetti S, Fish A, Whitley CB, Jaeger P, Gertner J, New M, DiBona FJ, Kaplan BS, Robertson G, Hendy GN, Fujiwara TM & Morgan K (1994). Nature and recurrence of AVPR2 mutations in X-linked nephrogenic diabetes insipidus. American Journal of Human Genetics, 55: 278-286.
  8. Bichet DG (1996). Vasopressin receptors in health and disease. Kidney International, 49: 1706-1711.
  9. Kanno K, Sasaki S, Hirata Y, Ishikawa S-E, Fushimi K, Nakanishi S, Bichet DG & Marumo F 1995). Urinary excretion of aquaporin-2 in patients with diabetes insipidus. New England Journal of Medicine, 332: 1540-1545.
  10. Deen PM, Verdijk MA, Knoers NV, Wieringa B, Monnens LA, van Os CH & van Oost BA (1994). Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science, 264: 92-95.

Departamento de Farmacologia, Universidade Federal de Minas Gerais, 31270-010 Belo Horizonte, MG, Brasil (J.L. Rocha, B.C. Silva, L. De-Marco), Departamento de Cliníca Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900 Ribeirão Preto, SP, Brasil (A.C. Moreira), Department of Hematology and Genetics, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel (E. Friedman), Unidade de Endocrinologia, Hospital Brigadeiro, 01401-901 São Paulo, SP, Brasil (B. Liberman)

April 1997

Address correspondence and reprint requests to Brazilian Journal of Medical and Biological Research

This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

X-linked nephrogenic diabetes insipidus (XNDI) is caused by mutations in the vasopressin-2 receptor gene that cause dysfunctional structural alterations in the vasopressin-2 receptor (V2R) that it helps synthesize. These structural alterations result in functional defects that usually do not permit the V2R to perform one of its functions. Many different mutations of the V2R gene that result in XNDI have been reported in the scientific literature. NDI may also be caused by mutations of the aquaporin-2 gene.

In this study, the authors analyzed a portion of the V2R gene called exon 3 in 20 unrelated individuals. (An exon is a segment of the gene that contains part of the code for the protein that the gene will help synthesize.) In nine of these individuals they found the same mutations. This mutation consisted of a thymine base (T) being where a cytosine base (C) usually is in a set of three bases called codon 331 of the V2R gene. However, this mutation did not alter the V2R amino acid the mutated gene helped produce. This mutation was found in seven healthy individuals with no symptoms of NDI and in two individuals with NDI.

This finding may indicate that possible structural variations in the sequence of a normal V2R gene are not accounted for in all cases of NDI. The authors suggest that the possible structural variations in the sequence of this V2R gene could be used to help better understand the pathophysiology of hereditary NDI.