Analysis of Vasopressin Receptor Type II (V2R) Gene in Three Japanese Pedigrees with Congenital Nephrogenic Diabetes Insipidus: Identification of a Family with Complete Deletion of the V2R Gene

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Title: Analysis of Vasopressin Receptor Type II (V2R) Gene in Three Japanese Pedigrees with Congenital Nephrogenic Diabetes Insipidus: Identification of a Family with Complete Deletion of the V2R Gene
Authors: Jinnouchi, MD, Hideaki; Araki, MD, Eiichi; Miyamura, MD, Nobuhiro; Kishikawa, MD, Hideki; Yoshimura, Ryohei; Isami, MD, Satoshi; Yamaguchi, MD, Kohei; Iwamatsu, MD, Hiroko; Shichiri, MD, Motoaki
Publisher: European Journal of Endocrinology
Date Published: June 01, 1996
Reference Number: 21
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To investigate the association of mutations in the arginine vasopressin receptor type II (V2R) gene with congenital nephrogenic diabetes insipidus (CNDI) in the Japanese, we analyzed the V2R gene, located on the X chromosome, in three Japanese pedigrees with CNDI. In one pedigree, a large deletion spanning the entire coding region of the V2R gene was identified. In another pedigree, a G to A transition responsible for a substitution of Met88 (ATG) for Val88 (GTG) was detected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that this was a de novo mutation that had occurred in the proband's mother. Because CNDI was observed only in those with this mutation, the pathogenicity of this mutation seemed clear. In the last pedigree, only a silent mutation of Leu309 (CTA-->CTG) was found. All the individuals studied in this pedigree by allele-specific oligonucloetide-polymerase chain reaction (ASO-PCR) analysis showed a complete association of this mutation to the clinical symptoms. Because the silent mutation detected was unlikely to be a direct cause of CNDI, mutations in other regions of the V2R gene, such as a promoter region or other regulatory regions, may be responsible for the cause of CNDI in this pedigree. Thus, association of the V2R gene abnormality to clinical symptoms of CNDI was confirmed in three Japanese pedigrees, and a strong contribution of the V2R gene mutation to the development of CNDI was suggested.
Reprinted by permission from the EUROPEAN JOURNAL OF ENDOCRINOLOGY, 1996 Jun;134(6):689-98 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. The copyright holder of this article is the Society of the European Journal of Endocrinology.


Congenital nephrogenic diabetes insipidus (CNDI) is a rare disease characterized by renal tubular insensitivity to the antidiuretic hormone arginine vasopressin (AVP) (1, 2). Polydipsia, polyuria and hyposthenuria are the main clinical characteristics of this disease, which is sometimes associated with mental retardation, physical retardation, hydronephrosis or renal failure.

Recently, signal transduction pathways for water resorption mediated by AVP in renal tubular cells have been characterized by the cloning of genes and cDNAs encoding for the signalling molecules, such as arginine vasopressin receptor type II (V2R) (3, 4) and aquaporins (AQP) (5-9). Linkage analysis showed that the gene responsible for CNDI had been localized to Xq28 (10, 11). In 1992 Birnbaumer et al. (3) cloned the human V2R cDNA (3) and human V2R gene. The human V2R gene is located in the q28-qter portion of the X chromosome (12), and lies on a 2.2-kb BamH I fragment containing three exons and two introns (3, 12). The sequence of V2R deduced from the cDNA revealed a polypeptide of 371 amino acids with seven transmembrane domains (3), which showed characteristics of members of the G-protein-coupled receptor superfamily (13). The V2R gene is mainly expressed in renal tubular cells (14). In response to increases in serum osmolality, AVP is secreted from the posterior pituitary gland and binds to receptors on the surface of renal tubular cells. Following AVP binding, V2R undergoes conformational changes that trigger receptor-G-protein interaction, facilitating an exchange of GTP for bound GDP at a site within the a-subunit of the trimeric G protein. Binding of GTP to the a-subunit of the G protein promotes dissociation of this subunit from the b- and g-subunits and results in activation of adenylate cyclase. Activated adenylate cyclase evokes an elevation of intracellular cyclic adenosine monophosphate (AMP) levels followed by stimulation of AMP-dependent protein kinase and by translocation of cytosolic endosomes embedded with water channels to the apical or basolateral surface membrane of collecting duct cells, which finally results in elevation of water permeability. Any defect in this signal transduction pathway for renal water resorption will lead to CNDI.

More than 50 mutations in the V2R gene, including missense mutations, non-sense mutations, frameshift mutations and large deletions in the 3'-region, have now been identified in CNDI patients, most of which are Caucasians and African-Americans, as summarized by Holtzman et al. (15). On the other hand, mutations of aquaporin 2 (AQP2) have also been identified in three CNDI patients (16, 17), thereby suggesting heterogeneity of CNDI.

In Japan, over 70 patients have been diagnosed with CNDI, approximately 30% of whom have a family history of the disease (18). However, only four cases of CNDI with V2R gene mutations, including two missense mutations, a single-base deletion and a three-base inframe deletion, have been documented (19, 20). Thus, it remained to be determined if other variants of V2R gene mutations or other causes of the disease could be identified in Japanese CNDI patients.

In this study, we have analyzed the V2R gene and assessed the association of mutations with the clinical symptoms of CNDI in three Japanese pedigrees with an apparent family history of CNDI.

Subjects and methods
Subjects and clinical data

Three Japanese pedigrees (pedigrees A-C in Fig. 1) with an apparent family history of congenital nephrogenic diabetes insipidus (CNDI) were studied. Clinical data were obtained from individuals who gave their informed consent. A water deprivation test and a 1-desamino-8-D-arginine vasopressin (dDAVP) loading test were performed as described previously (21, 22). Serum AVP concentration was determined using a radioimmunoassay (23). Plasma and urine osmolality were measured cryoscopically (24).

Amplification of the V2R gene using the polymerase chain reaction (PCR)

Genomic DNA from peripheral blood leukocytes from the probands and their relatives was isolated as described elsewhere (25). To amplify the V2R gene, primers were designed from the human V2R gene sequence (3, 12) as shown in Fig. 2. PCR amplification was carried out using 1 mg of genomic DNA, 100 pmol of each primer, 2.5 mmol/l of dNTPs and 2.5 U of Ampli Taq DNA polymerase (Perkin Elmer/Cetus Norwalk, CT) in 100 ml of reaction buffer (10 mmol/l TRIS (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl2 and 0.001% gelatin). The reaction was controlled using a thermal cycle programmer (Model PC-700; Astec, Fukuoka, Japan) under denaturation conditions of 95°C for 5 min and annealing at 58°C for 3 min for one cycle, subsequent extension at 74°C for 1 min. denaturation at 95°C for 1 min and annealing at 58°C for 30s for 35 cycles, and finally extension at 74°C for 10 min for one cycle (26). The PCR products were separated on a 1.5% agarose gel and visualized using ethidium bromide staining.

Southern blot analysis of PCR products

After agarose gel electrophoresis of the PCR products, the gel was soaked once in denaturing buffer (1.5 mol/l NaCl/0.5 mol/I NaOH) for 30 min and twice in neutralization buffer (3 mol/l NaCl/0.5 mol/l TRIS · HCl) for 15 min, and the PCR products were transferred onto a nitrocellulose filter membrane and fixed by baking at 80°C for 2 h. The filter was then prehybridized for 6 h at 42°C and hybridized with a labeled probe (1.45 x 109 cpm/mg). A 1719-bp PCR product that covers the entire coding region and the first and second introns of the V2R gene (nt —52 to +1193 in Ref. 3) was amplified from genomic DNA of a normal control (male), using primers A and H (shown in Fig. 2). This fragment was purified and subcloned into the pUC19 plasmid and used as a probe after confirming the sequence. After hybridization, the filter was washed for 2 h twice with 0.1 x SSC/0.1% SDS at 55°C and subjected to autoradiography.

Southern blot analysis of genomic DNA

Genomic DNA (5 mg) from each individual was digested with endonuclease EcoR I, Nco I and Pst I, respectively, in 20 ml of reaction mixture and electrophoresed in a 0.8% agarose gel at 50 mV for 8 h. After electrophoresis, the separated DNA was transferred onto a nitrocellulose filter, as described previously. Two probes corresponding to the V2R gene and the a-galactosidase cDNA, both of which are located on the X chromosome, were used for analysis. Human a-galactosidase cDNA (27, 28) was synthesized from RNA prepared from human fibroblasts by the reverse transcriptase-PCR method (29) using two primers (5'-ATGCAGCTGAGGAACCCA-3' and 5'-TGACATCTGCATTGTATT-3': nt 23-40 and 1292-1275, respectively, in Ref. 27) and subcloned into the pUC19 plasmid. Each probe was labeled using the Megaprime DNA labeling system (Amersham International, UK). The same filter was hybridized with the V2R gene probe only (Fig. 4B, 4D) and with both the V2R gene and the a-galactosidase cDNA probes (Fig. 4C), as described previously.

Deoxyribonucleic acid sequencing

For sequence analysis of the V2R gene in probands B and C, the entire coding region and the first and second introns of the V2R gene (nt —52 to 1193) were amplified by PCR using either a set of primer pair A and H or four sets of primer pairs shown in Fig. 2. The PCR product was then separated on a 2% agarose gel, purified and subcloned in the plasmid vector. The sequence was then determined using an ABI 373A autosequencer (Applied Biosystems) and the dideoxynucleotide method using Sequenase version 2.0 sequencing kits (United States Biochemical Corp., Cleveland, OH) (30).

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific oligonucleotide (ASO)-PCR analysis

Sequence analysis revealed point mutations in the V2R gene in probands B and C at different positions. To assess the association of each mutation with the clinical symptoms in each pedigree, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of pedigree B and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) analysis of pedigree C were performed.

For PCR-RFLP analysis in pedigree B, a 505-bp fragment (nt 33-537), including the sequence in which the point mutation was found in proband B, was amplified from genomic DNA by PCR, using a primer pair of C and D (Fig. 2). Because the mutation found in a proband B created a novel Nco I site at nucleotide position 262, the PCR product was digested with endonuclease Nco I to assess existence of the mutation. Samples were then separated on a 4% NuSieve 3: 1 agarose gel (FMC Corp., Rockland, ME) and visualized using ethidium bromide staining.

For ASO-PCR analysis in pedigree C, we designed two antisense primers, one of which corresponds to the sequence of the wild-type V2R gene (5'-GGCCAGCAA-CATGAGTA-3') and the other to that of the mutant V2R gene found in proband C (5'-GGCCAGCAACATGAGCA-3'). Primer E was used as a sense primer in both reactions. The PCR reaction was performed as described previously except that annealing was carried out at 65?C instead of 58?C. The PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining.

Results
Pedigrees

The pedigrees of three kindreds are shown in Fig. 1 and clinical data of the individuals studied are listed in Table 1.

Pedigree A. The proband (proband A) was a 38-year-old man who had complained of severe polydipsia and polyuria since childhood. The volume of urine excreted was over 101 per day and urine osmolality was under 200 momsmol/kg. Lack of change in urine osmolality following a water deprivation test and a dDAVP loading test (21, 22) led to a diagnosis of nephrogenic diabetes insipidus (Table 1). Severe hydronephrosis and moderate renal failure were present, but there was no evidence of mental or physical retardation. The proband's younger brother (III-3 in Fig. 1A) was also diagnosed with CNDI, based on a dDAVP loading test (Table 1). One of two daughters of the proband (IV-2 in Fig. 1A) had severe polydipsia and polyuria, and showed significant hyposthenuria and a high serum ADH concentration, at similar levels to those of the proband (Table 1). The clinical data of the proband's mother and another daughter (II-2 and IV-1 in Table 1, respectively) were normal. In this pedigree, the siblings of the proband's maternal grandmother and her cousin also complained of polydipsia and polyuria (I-3, I-4 and II-5, respectively, in Fig. 1A). No individual was found to show polydipsia and polyuria in the proband's wife's family (data not shown). Because no male to male transmission was observed, X-linked inheritance for CNDI was suggested for this pedigree.
Fig. 1. Pedigrees of the three families with congenital nephrogenic diabetes insipidus (CNDI) studied. Pedigrees of the three CNDI families (pedigrees A, B and C) are shown in A, B and C, respectively. Individuals diagnosed as CNDI on the basis of insensitivity to vasopressin analog are indicated by filled symbols. Individuals with polydipsia and polyuria whose urine was confirmed to be under 200 mosmol/kg are indicated by shaded symbols. Individuals reported to have polydipsia and polyuria are indicated by half-shaded symbols. Individuals without polydipsia or polyuria are indicated by open symbols. Each proband is indicated by an arrow.

Table 1. Clinical data of members in the pedigrees studied.

Deprivation test
u-Osm
(mosm/kg)

dDAVP test
u-Osm
(mosm/kg)

s-ADH
pg/ml)
s-Osm
(mosm/kg)
Random
u-Osm
(mosm/kg)
Daily
u-Volume
(ml/kg/day)
Before
3h
Before
1h

Pedigree A
II-2
2.7
278
447
31.3
--
--
--
--
III-Ia
14.4
298
121
105.6
84
81
82
76
III-3
9.2
299
101
110.7
106
95
91
82
IV-1
3.0
281
594
19.0
--
--
--
--
IV-2
8.8
293
102
113.0
--
--
--
--
 
Pedigree B
II-2
1.0
289
727
28.5
--
--
--
--
II-4
1.4
290
894
18.7
--
--
--
--
III-1
1.5
287
405
29.8
--
--
--
--
III-3
3.7
291
58
175.1
58
97
92
138
III-4
3.0
290
1167
22.3
--
--
--
--
IV-1
5.7
278

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)

This article reports on the investigations of Jinnouchi, et.al., into the association of mutations in the arginine vasopressin receptor type II gene (V2R) with congenital nephrogenic diabetes insipidus (CNDI). The authors analyzed the V2R gene located on the X chromosome in four generations of three unrelated Japanese families where there was a confirmed case of CNDI. The individual with the confirmed CNDI is referred to as a proband and serves as a starting point to track the occurrence of CNDI in the proband's generation as well as generations preceding and succeeding it. There is a proband for family A, for family B, and for family C.

CNDI is characterized by the kidneys' insensitivity to the antidiuretic hormone, arginine vasopressin (AVP). This results in the kidneys being unable to reabsorb water for the body's use and instead voiding it all through urination. The main symptoms of CNDI are extreme thirst, excessive urination and high sodium levels in the blood. CNDI is sometimes associated with mental and/or physical retardation, hydronephrosis, or kidney failure.

Genes and chromosomes are microscopic, of course, but researchers have been able to, in effect, enlarge them to the degree where a single gene becomes like a building in the neighborhood of the chromosomes. The researcher is like a building inspector taking note of the building to see if it is properly constructed and functions as it should. The human V2R gene is located in a portion of the X chromosomes called the q28-qter. Its sequence, or architecture in this analogy, consists of a chain of 371 amino acids with seven transmembrane domains. The V2R gene works mainly in the tubular cells in the kidneys where, if all is working well, it accepts the information from the antidiuretic hormone, AVP. This allows an intricate sequence to take place, which results in an elevation of cellular water permeability, which allows the kidneys to reabsorb water for the body's use. Any defect in this sequence, which hinges on proper functioning of the V2R receptor, will lead to CNDI.

Researchers know that mutations in the V2R gene will render it incapable of doing its job. They also know more than 50 different types of mutations of the V2R gene have been identified. In some, called nonsense mutations, there is a sequence misplacement. For example, a part of the genetic material that should be at the end of a genetic message instead appears in the middle of the message. Another type of mutation, called a missense mutation, occurs when parts of the material that make up the gene are altered so that different amino acids are produced than should be. And some mutations involve portions of the genetic material simply not being present. An added complexity is that a few cases of CNDI have been associated, not with mutations of the V2R gene, but with mutations in a water transporting protein called an aquaporin 2 (AQP2).

In this study, the authors found the mutations associated with CNDI in each of the three extended Japanese families were all V2R mutations, but each was a different type of mutation.

In the lineage of family A, the proband was a 38-year-old man. His younger brother was also diagnosed with CNDI. One of the proband's daughters showed signs of CDNI: excessive thirst and urination, urine with a low specific gravity, and a high serum ADH concentration. In proband A's family tree, his maternal grandmother's brother and sister also complained of excessive thirst and urination. Because no male-to-male transmission was observed, the researchers concluded that the CNDI was an X-linked inheritance.

In family B, the proband was an 8-year-old boy. His mother and elder brother were diagnosed by the researchers as also having CNDI. The family C proband was a 3-year-old boy. In this family tree, four other males and two females were diagnosed with CNDI, and two males and four females complained of CNDI symptoms, though clinical data was not obtained on them. Again, no male-to-male transmission was observed, suggesting X-linked inheritance of CNDI.

To determine the nature of the V2R gene mutation in probands A, B and C, extensive analyses were undertaken. The author's found the mutation in proband A was a large deletion spanning the entire coding region of the V2R gene. The coding region of the V2R gene simply wasn't in proband A's genetic structure. Neither was it in his brother's. To date, all other reported V2R mutations have been either point mutations, insertions, or deletions of several parts of the gene called bases, or they have been a large deletion affecting a specific region of the gene. Proband A's deletion is apparently the first known mutation that has resulted in complete deletion of the coding region of the V2R gene. Sometimes when an entire gene is deleted, something else will assume its function. This was not the case for Proband A. This suggests that there is no major pathway other than the V2R gene that can transduce the signal of AVP to promote water reabsorption in the kidneys' tubular cells.

The mutation in proband B's V2R gene was a missense mutation -- a part of the gene labeled "A" was where a part of the gene labeled "G" was supposed to be. This created the amino acid Met88 (ATG) where amino acid Val88 (GTG) should have been. This mutation resulted in creation of a novel Neo 1 restriction site at this position. This same mutation was found in the proband's mother and brother. Since this mutation was not found in other members of the family tree, the researchers concluded that this was a new mutation that had occurred in proband B's mother and passed on to her two sons. The fact that this mutation occurred first in proband B's mother, not before, and was associated with CNDI, suggests to the authors that this mutation caused the CNDI.

Proband C had a silent mutation at Leu309 (CTA to CTG) that resulted in no amino acid change. There was a strong association with this mutation to clinical symptoms of CNDI, but since the mutation does not seem to disrupt either the donor or acceptor sites which are important for maturation of mRNA (a message from gene to amino acid sequience), it does not seem to be a direct cause of the disease. But since all three mutations were associated with clinical symptoms of CNDI in all three Japanese family trees, the importance of the V2R gene in the pathogenesis of CNDI in Japanese patients was confirmed.