X-linked Nephrogenic Diabetes Insipidus Research Articles

The ins and outs of aquaporin-2 trafficking

This review outlines recent advances related to the molecular mechanisms and pathways of aquaporin-2 (AQP2) water channel trafficking. AQP2 is a fascinating protein, whose sorting signals can be interpreted by different cell types to achieve apical or basolateral membrane insertion, in both regulated and constitutive trafficking pathways. In addition to the well-known cAMP-mediated, stimulatory effect of vasopressin on AQP2 membrane insertion, other signaling and trafficking events can also lead to AQP2 membrane accumulation via cAMP-independent mechanisms. These include 1) elevation of cGMP, mediated by sodium nitroprusside (a nitric oxide donor), atrial natriuretic factor, and l-arginine (via nitric oxide synthase); 2) disruption of the actin cytoskeleton; and 3) inhibition of the clathrin-mediated endocytotic arm of the AQP2 recycling pathway by dominant-negative dynamin expression and by membrane cholesterol depletion. Recent data also indicate that AQP2 recycles constitutively in epithelial cells, it can be inserted into different membrane domains in different cell types both in vitro and in vivo, and these pathways can be modulated by factors including hypertonicity. The roles of accessory proteins, including small GTPases and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins in AQP2 membrane insertion, are also being uncovered. Understanding cAMP-independent mechanisms for membrane insertion of AQP2 is especially relevant to the therapeutic bypassing of the mutated, dysfunctional vasopressin receptor in patients with X-linked nephrogenic diabetes insipidus.

Aminoglycoside pretreatment partially restores the function of truncated V(2) vasopressin receptors found in patients with nephrogenic diabetes insipidus.

By screening patients with X-linked nephrogenic diabetes insipidus (NDI) for mutations within the V(2) vasopressin receptor (AVPR2) gene, we have identified six novel and two recurrent mutations. Additionally, one patient revealed a genomic deletion of 3.2 kb encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene, which is in close proximity to the AVPR2 locus. In-depth characterization of the mutant AVPR2s by a combination of functional and immunological techniques allowed to gain further insight into molecular mechanisms leading to the receptor dysfunction. Aiming at the functional reconstitution of mutant G protein-coupled receptors, several strategies of potential therapeutic usefulness have been tested. Because the functional rescue of truncated receptors is most challenging, we addressed this issue by applying an aminoglycoside approach. Here, we demonstrate that the misreading capacity of the aminoglycoside antibiotic geneticin was sufficient to restore function of mutant AVPR2s harboring premature stop codons in an in vitro expression system.

Intrafamilial phenotype variability in nephrogenic diabetes insipidus

X-Linked nephrogenic diabetes insipidus (NDI), which accounts for 90% of inherited cases of NDI, is caused by mutations in the AVPR2 gene that encodes the arginine vasopressin (AVP) receptor type 2 (V2R). The V2R mediates the antidiuretic action of AVP in principal cells of the collecting duct. To date, only three AVPR2 mutations (P322S, D85N, and G201D) have been associated with a mild NDI phenotype, and intrafamilial phenotype variability has not been reported in affected males. We describe a novel Belgian family with X-linked NDI caused by substitution of a histidine for an arginine at position 137 (R137H) of AVPR2. This mutation has been identified in two brothers and their mother. The R137H mutation results in a failure of V2R to stimulate adenylate cyclase and has been associated consistently with severe NDI and the inability to increase urinary osmolality to greater than plasma osmolality during water deprivation and/or infusion of 1-desamino-8-d-arginine vasopressin. Detailed examination of the two affected brothers showed the typical NDI phenotype in the 45-year-old proband, whereas a milder clinical phenotype associated with significant urinary concentrating ability during water deprivation was documented in the 33-year-old brother. Thus, in this family, the R137H mutation is associated with either a mild or severe NDI phenotype. Mechanisms that might account for these findings include genetic and/or environmental modifiers. © 2002 by the National Kidney Foundation, Inc.

Functional Rescue of the Nephrogenic Diabetes Insipidus-causing Vasopressin V2 Receptor Mutants G185C and R202C by a Second Site Suppressor Mutation

Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.

Generation and phenotype of mice harboring a nonsense mutation in the V2 vasopressin receptor gene

The V2 vasopressin receptor (V2R) plays a key role in the maintenance of a normal body water balance. To generate an in vivo model that allows the physiological and molecular analysis of the role of V2Rs in kidney function, we have created mouse lines that lack functional V2Rs by using targeted mutagenesis in mouse embryonic stem cells. Specifically, we introduced a nonsense mutation known to cause X-linked nephrogenic diabetes insipidus (XNDI) in humans (Glu242stop) into the mouse genome. V2R-deficient hemizygous male pups showed a decrease in basal urine osmolalities and were unable to concentrate their urine. These pups also exhibited an enlargement of renal pelvic space, failed to thrive, and died within the first week after birth due to hypernatremic dehydration. Interestingly, female mice heterozygous for the V2R mutation showed normal growth but displayed an XNDI-like phenotype, characterized by reduced urine concentrating ability of the kidney, polyuria, and polydipsia. Western blot analysis and immunoelectron microscopic studies showed that the loss of functional V2Rs had no significant effect on the basal expression levels of aquaporin-2 and the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1). The V2R mutant mice described here should serve as highly useful tools for the development of novel therapeutic strategies for the treatment of XNDI.

A novel splicing mutation in the V 2 vasopressin receptor

In order to elucidate the molecular basis and the clinical characteristics of X-linked recessive nephrogenic diabetes insipidus (CNDI) in a kindred of Danish descent, we performed direct sequencing of the arginine vasopressin receptor 2 (AVPR2) gene in five members of the family, as well as clinical investigations comprising a fluid deprivation test and a 1-deamino-8-D-arginine-vasopressin (dDAVP) infusion test in the study subject and his mother. We found a highly unusual, novel, de novo 1447A-->C point mutation (gDNA), involving the invariable splice acceptor of the second intron of the gene in both the affected male (hemizygous) and his mother (heterozygous). This mutation is likely to cause aberrant splicing of the terminal intron of the gene, leading to a non-functional AVP receptor. The clinical studies were consistent with such a hypothesis, as the affected subject had a severe insensitivity to both the antidiuretic and the coagulation factors stimulatory actions of AVP and its analogue dDAVP. Direct sequencing of the AVPR2 is an accurate and rapid diagnostic tool for CNDI and early referral of patients for AVPR2 sequencing is therefore strongly suggested.

Report of 33 novel AVPR2 mutations and analysis of 117 families with X-linked nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease caused by mutations in the arginine vasopressin receptor 2 gene (AVPR2). Thirty-three novel AVPR2 mutations were identified in 62 families that were not included in our previous studies. This study describes the diversity of mutations observed in a total of 117 families, the number of affected people at the time of diagnosis, skewed X chromosome inactivation in severely affected females, the inferred parental origin of de novo mutations, and it provides estimates of incidence. Among 117 families, there were 82 different putative disease-causing mutations. Based on haplotype analysis, it can be inferred that when the same AVPR2 mutation is identified in different families that were not known to be related, the mutations most likely arose independently. More than half of the families had only one affected male; two families presented with a severely affected female and no family history of NDI. A de novo mutation arose during oogenesis in the mother in 20% of isolated cases. The estimate of about 8.8 per million male live births of the incidence of X-linked NDI in the province of Quebec, Canada may be representative of the general population except in Nova Scotia and New Brunswick, where the incidence is more than six times higher. Documentation of the diversity of mutations will assist in revealing the full spectrum of clinical variation. Discussion of genetic and population genetic aspects of X-linked NDI may contribute to early diagnosis and treatment.

Nephrogenic diabetes insipidus: functional analysis of new AVPR2 mutations identified in Italian families.

The aim of this study was to identify loss-of-function mutations of the V2 vasopressin receptor gene (AVPR2) in Italian patients affected by X-linked nephrogenic diabetes insipidus (NDI). Mutations were found in 15 of the 18 unrelated families investigated: nine of these mutations were previously unknown, including two affecting residues located in regions known to be important for determining the pharmacologic properties of the receptor, which were therefore functionally investigated. The first (A84D) involves a residue located near an aspartic acid (D85) that is highly conserved in all G protein-coupled receptors and that is believed to play a role in the process of their isomerization into functionally active and inactive states. The present study indicates that this mutation not only affects receptor folding in such a way as to lead to its retention inside the intracellular compartments but, as expected, also has profound effects on its binding and coupling properties. The second was a mutation of a tryptophan located at the beginning of the first extracellular loop (W99R) that greatly impaired the binding properties of the receptor and had a minor effect on its intracellular routing. Molecular analysis of the first extracellular loop bearing this mutation suggests that this residue plays a fundamental role in stabilizing the peptide/receptor interactions responsible for the high-affinity binding of agonists to the V2 receptor.

Diabetes insipidus.

Diabetes insipidus.

Functional characterization of the molecular defects causing nephrogenic diabetes insipidus in eight families.

X-Linked nephrogenic diabetes insipidus (NDI) is a rare inherited disorder characterized by the excretion of abnormal large volumes of diluted urine mainly caused by mutations in the V2 vasopressin receptor (AVPR2) gene. By screening NDI patients for mutations within the AVPR2 gene we have identified three novel (I46K, F105V, I130F) and four recurrent (D85N, R106C, R113W, Q225X) mutations. In addition, a recurrent missense mutation (A147T) within the aquaporin-2 gene was identified in a female patient with autosomal recessive NDI associated with sensorineural deafness. Selected clinical data of the NDI patients were compared with the results from the in vitro studies. Functional analysis of I46K and I130F revealed reduced maximum agonist-induced cAMP responses as a result of an improper cell surface targeting. In contrast, the F105V mutation is delivered to the cell surface and displayed an unchanged maximum cAMP response, but impaired ligand binding abilities of F105V were reflected in a shifted concentration-response curve toward higher vasopressin concentrations. As the extracellularly located F105 is highly conserved among the vasopressin/oxytocin receptor family, functional analysis of this residue implicates an important role in high affinity agonist binding.

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell’s interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC 50 value and with a reduced EC max, indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.

Polarized expression of the vasopressin V2 receptor in Madin-Darby canine kidney cells

The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented. Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [3H]-arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti-c-myc antibody. The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell. The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor.

Compound deletion of the rhoGAP C1 and V2 vasopressin receptor genes in a patient with nephrogenic diabetes insipidus

The function of small GTPases is fine-tuned by a complex network of regulatory proteins such as GTPase-activating proteins. The C1 gene at Xq28 encodes a protein assumed to function as a Rho GTPase-activating protein (rhoGAP). Characterization of the molecular defect causing X-linked nephrogenic diabetes insipidus (NDI) in a patient revealed a submicroscopic deletion of a 21.5-kb genomic fragment encompassing the entire arginine-vasopressin V2 receptor gene (AVPR2) and most of the C1 gene locus. In the absence of detailed information about the physiological relevance and specific functions of rhoGAP C1, a thorough clinical and laboratory investigation of the patient was performed. Besides clearly defined NDI symptoms caused by deletion of the AVPR2 gene, no major morphological abnormalities as determined by physical examination, radiography, ultrasound, and computed tomographic scan were detected. Extensive analysis of blood chemical, enzyme, and hormone values over a period of 16 years showed no deviations from normal ranges. On the basis of our observations, the rhoGAP C1 protein is not essential for normal development in the human. Because of a predominant expression pattern of the C1 gene in hematopoietic cells, we focused on immunologic and hematologic laboratory parameters of the affected boy and the mother who was found to be heterozygous. Differential white cell counts, including lymphocyte typing, determination of lymphokines, cytokines, and immunoglobulins, as well as numerous leukocyte function tests, showed no pathological findings. Therefore, we postulate that the loss of rhoGAP C1 function is most likely compensated by other members of the GAP family. Hum Mutat 14:163–174, 1999. © 1999 Wiley-Liss, Inc.

Molecular analyses of the vasopressin type 2 receptor and aquaporin-2 genes in Brazilian kindreds with nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is associated with germline mutations in two genes: vasopressin receptor type 2 (V2(R)) in X-linked NDI, and the water channel aquaporin-2, in autosomal-recessive disease. Genetic heterogeneity is further emphasized by reports of phenotypically abnormal individuals with normal structural genes. We analyzed both genes in five Brazilian families and the aquaporin-2 gene in two Swedish families with clinical and laboratory diagnosis of NDI, by a combination of denaturing gradient gel electrophoresis (DGGE) and direct DNA sequencing. A novel polymorphism in the aquaporin-2 gene (S167S), but no disease-associated mutations in any tested individual from all seven families, was detected. In two Brazilian families, frameshift mutations were detected in the V2(R) gene: one leading to a premature stop after codon 36 and the other to a longer peptide (462 aa instead of the 373 aa wild-type protein). In two other Brazilian families, probable disease-associated missense mutations were detected: an alanine to proline at codon 163 (A163P) and an asparagine to aspartic acid at codon 85 (D85N). In one Brazilian family, both genes were structurally normal and the aquaporin-2 gene was also normal in the two Swedish kindreds. This report further extends the mutational spectrum of NDI and suggests that there are other mutational or epigenetic events inactivating the two known genes or even novel genes that underlie NDI.

Molecular analyses of the vasopressin type 2 receptor and aquaporin-2 genes in Brazilian kindreds with nephrogenic diabetes insipidus

Nephrogenic diabetes insipidus (NDI) is associated with germline mutations in two genes: vasopressin receptor type 2 (V2(R)) in X-linked NDI, and the water channel aquaporin-2, in autosomal-recessive disease. Genetic heterogeneity is further emphasized by reports of phenotypically abnormal individuals with normal structural genes. We analyzed both genes in five Brazilian families and the aquaporin-2 gene in two Swedish families with clinical and laboratory diagnosis of NDI, by a combination of denaturing gradient gel electrophoresis (DGGE) and direct DNA sequencing. A novel polymorphism in the aquaporin-2 gene (S167S), but no disease-associated mutations in any tested individual from all seven families, was detected. In two Brazilian families, frameshift mutations were detected in the V2(R) gene: one leading to a premature stop after codon 36 and the other to a longer peptide (462 aa instead of the 373 aa wild-type protein). In two other Brazilian families, probable disease-associated missense mutations were detected: an alanine to proline at codon 163 (A163P) and an asparagine to aspartic acid at codon 85 (D85N). In one Brazilian family, both genes were structurally normal and the aquaporin-2 gene was also normal in the two Swedish kindreds. This report further extends the mutational spectrum of NDI and suggests that there are other mutational or epigenetic events inactivating the two known genes or even novel genes that underlie NDI. Hum Mutat 14:233–239, 1999. © 1999 Wiley-Liss, Inc.

Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. To study the cause of loss of function of mutant V2 receptors, we expressed 12 mutations (N55H, L59P, L83Q, V88M, 497CC-->GG, deltaR202, I209F, 700delC, 908insT, A294P, P322H, P322S) in COS-7 cells. Eleven of these, including P322H, were characterized by a complete loss of function, but the mutation P322S demonstrated a mild clinical and in vitro phenotype. This was characterized by a late diagnosis without any growth or developmental delay and a significant increase in urine osmolality after intravenous 1-deamino[D-Arg8]AVP administration. In vitro, the P322S mutant was able to partially activate the Gs/adenylyl cyclase system in contrast to the other V2R mutants including P322H, which were completely inactive in this regard. This showed not only that Pro 322 is important for proper V2R coupling, but also that the degree of impairment is strongly dependent on the identity of the substituting amino acid. Three-dimensional modeling of the P322H and P322S mutant receptors suggested that the complete loss of function of the P322H receptor could be due, in part, to hydrogen bond formation between the His 322 side chain and the carboxyl group of Asp 85, which does not occur in the P322S receptor.

The molecular basis of nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is characterized by resistance of the kidney to the action of arginine-vasopressin (AVP); it may be due to genetic or acquired causes. Recent advances in molecular genetics have allowed the identification of the genes involved in congenital NDI. While inactivating mutations of the vasopressin V2 receptor are responsible for X-linked NDI, autosomal recessive NDI is caused by inactivating mutations of the vasopressin-regulated water channel aquaporin-2 (AQP-2). About 70 different mutations of the V2 receptor have been reported, most of them missense mutations. The functionally characterized mutants show a loss of function due to defects in their synthesis, processing, intracellular transport, AVP binding, or interaction with the G protein/adenylyl cyclase system. Thirteen different mutations of the AQP-2 gene have been reported. Functional studies of three AQP-2 mutations reveal impaired cellular routing as the main defect. The great number of different mutations with various functional defects hinders the development of a specific therapy. Gene therapy may, however, eventually become applicable to the congenital forms of NDI. At present all gene-therapeutic approaches lack safety and efficiency, which is of particular relevance in a disease that is treatable by an adequate water intake. The progress with regard to the molecular basis of antidiuresis contributes to the understanding of acquired forms of NDI on a molecular level. Recent data show that lithium dramatically reduces the expression of AQP-2. Likewise, hypokalemia reduces the expression of this water channel. The exact mechanisms leading to this reduced expression of AQP-2 remain to be determined.

Functional study of two V2 vasopressin mutant receptors related to NDI. P322S and P322H.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine-vasopressin (AVP) V2 receptor response due to mutations in the AVPR2 gene in Xq2 (1). Most of the mutations already described are characterized by a complete loss of function, but some of them are of special interest as the patients harboring those mutant V2R have a mild clinical phenotype. We report here the study of two mutations of the same proline residue at position 322 (residue 22 in the transmembrane domain seven) in the V2R: P322S and P322H, characterized by a mild, i.e. a response in urinary osmolality, albeit mild, observed on V2R selective agonist administration, and a complete phenotype respectively.

A novel mutation in the neonatal region of the fibrillin (FBN)1 gene associated with a classical phenotype of Marfan syndrome (MfS). Mutations in brief no. 163. Online.

Marfan Syndrome (MfS) is an autosomal dominant inherited connective tissue disorder with variable phenotypic expression of cardiovascular, skeletal and ocular manifestations. Cardiovascular complications, such as aortic aneurysm and dissection drastically reduce life expectancy of individuals with MfS, whereas preventive surgery substantially improves the prognosis of these patients. A number of mutations in the fibrillin 1 (FBN1) gene associated with MfS have been identified to date, demonstrating considerable molecular heterogeneity. One region, however, located around exon 24, exhibits a striking clustering of mutations, which are associated with a severe, socalled neonatal form of MfS. Here we report the first mutation (G2950A) in exon 24 of the neonatal region of the FBN1 gene, associated with a classic MfS phenotype. The mutation leads to the subsitution of valin by isoleucin (V984I), both uncharged amino acids, which only differ in a single methyl group. This defect was identified in a proband with cardiovascular manifestations of MfS by SSCP analysis of PCR-amplified genomic DNA, direct PCR sequencing and RFLP analysis. The substitution was neither detected in the unaffected 4-year old daughter of the proband, nor in 3 of his healthy family members nor in 108 allels from control individuals, suggesting that this mutation is causative for MfS in the patient. Since no other family member of the proband is affected by MfS, the defect described is sporadic. In summary, we identified a novel defect in exon 24 of the neonatal region of the FBN1 gene in a patient with a classic phenotype of MfS, suggesting that conservative substitutions in this region may lead to a less severe phenotype of the disease. This finding further demonstrates the remarkable phenotypic heterogeneity associated with FBN1 mutations and stresses the significance of modifying genes and individual alterations in protein function for the pheontypic expression of the disease.

C112R, W323S, N317K mutations in the vasopressin V2 receptor gene in patients with nephrogenic diabetes insipidus

Nephrogenic diabetes insipidus (NDI) is a rare, mostly X-linked recessive disorder characterized by renal tubular resistance to the antidiuretic effect of arginine vasopressin. The gene responsible for the X-linked NDI, the G-protein-coupled vasopressin V2 receptor, has been localized on the Xq28 region. In this study we present three NDI families from Hungary with three different missense mutations in the vasopressin V2 receptor gene. After the mutations in the affected probands in each family had been characterized, other family members were screened by restriction enzyme analysis. The N317K and the W323S mutations have not been detected previously. The C112R is an already known mutation. The N317K was a de novo mutation in the patient. The C112R and the W323S were found in the mothers of the patients as carriers and in all other patients, but not in the unaffected members of the families. Segregation of the mutations was consistent with the clinically observed symptoms as well as their severity. As conclusion, these findings provide further evidence that X-linked NDI results from defects in the V2 receptor gene. Hum Mutat 12:137–138, 1998. © 1998 Wiley-Liss, Inc.