Identification of mutations in the arginine vasopressin receptor 2 gene causing nephrogenic diabetes insipidus in Chinese patients

Congenital nephrogenic diabetes insipidus (NDI) is, in most instances, a rare X-linked recessive renal disorder (MIM 304800) characterized by the clinical symptoms of polyuria, polydipsia, and dehydration. The X-linked NDI is associated with mutations of the arginine vasopressin receptor type 2 (AVPR2) gene, which results in resistance to the antidiuretic action of arginine vasopressin (AVP) in the renal tubules and collecting ducts. Identification of mutations in the AVPR2 gene can facilitate early diagnosis of NDI, which can prevent serious complications such as growth retardation and mental retardation. We analyzed three unrelated Chinese NDI families and identified three mutations: R106C, F287L, and R337X. In addition, an A/G polymorphism at cDNA nucleotide position 927 (codon 309L) was identified. A functional expression assay of the R106C and F287L mutants in COS-7 cells revealed that both mutants show significant dysfunction and accumulate intracellular cyclic adenosine monophosphate in response to AVP hormone stimulation. These results facilitate the diagnosis of NDI at the molecular level in the Chinese population, and provide insight into the molecular pathology of NDI. Received: July 4, 2001 / Accepted: December 4, 2001     

The proteasome is involved in the degradation of different aquaporin-2 mutants causing nephrogenic diabetes insipidus

Mutations in the water channel aquaporin-2 (AQP2) can cause congenital nephrogenic diabetes insipidus. To reveal the possible involvement of the protein quality control system in processing AQP2 mutants, we created an in vitro system of clone 9 hepatocytes stably expressing endoplasmic reticulum-retained T126M AQP2 and misrouted E258K AQP2 as well as wild-type AQP2 and studied their biosynthesis, degradation, and intracellular distribution. Mutant and wild-type AQP2 were synthesized as 29-kd nonglycosylated and 32-kd core-glycosylated forms in the endoplasmic reticulum. The wild-type AQP2 had a t(1/2) of 4.6 hours. Remarkable differences in the degradation kinetics were observed for the glycosylated and nonglycosylated T126M AQP2 (t(1/2) = 2.0 hours versus 0.9 hours). Moreover, their degradation was depending on proteasomal activity as demonstrated in inhibition studies. Degradation of E258K AQP2 also occurred rapidly (t(1/2) = 1.8 hours) but in a proteasome- and lysosome-dependent manner. By triple confocal immunofluorescence microscopy misrouting of E258K to lysosomes via the Golgi apparatus could be demonstrated. Notwithstanding the differences in degradation kinetics and subcellular distribution such as endoplasmic reticulum-retention and misrouting to lysosomes, both T126M and E258K AQP2 were efficiently degraded. This implies the involvement of different protein quality control processes in the processing of these AQP2 mutants.     

Functionality of aquaporin-2 missense mutants in recessive nephrogenic diabetes insipidus

Aquaporin-2 (AQP2) missense mutants in recessive nephrogenic diabetes insipidus (NDI) are all retained in the endoplasmic reticulum (ER), but some could function as water channels. No conclusions could be drawn about the water permeability (Pf) of others, because there was no method for quantifying AQP2 expression in the plasma membrane. We recently developed such a method, which has allowed us to study the functionality of these AQP2 mutants. Immunoblot analysis of membranes of injected oocytes revealed that all mutants (AQP2-G64R, AQP2-N68S, AQP2 T126M, AQP2-A147T, AQP2-R187C, AQP2-S216P) are expressed as unglycosylated and high-mannose glycosylated AQP2. The level of the high-mannose form of AQP2-A147T in the plasma membranes was low, indicating that this mutation has a less severe effect on proper folding. Analysis of Pf values and plasma membrane expression levels reveals that AQP2-N68S, AQP2-R187C and AQP2-S216P are non-functional, AQP2-A147T is as functional as wt-AQP2, while AQP2-T126M and AQP2-G64R retain 20% of the permeability of wt-AQP2. Since G64 is highly conserved between AQPs and expected to form essential interactions with other amino acids within AQP1, the residual functionality of AQP2-G64R is surprising. Our data furthermore indicate that an eventual therapy with chemical chaperones that restores the routing of AQP2 mutants to the apical membrane of collecting ducts cells might relieve NDI in patients encoding AQP2-A147T, and to a lesser extent AQP2-T126M and AQP2-G64R, but not in patients encoding AQP2-N68S, AQP2-R187C or AQP2-S216P.     

V2 vasopressin receptor dysfunction in nephrogenic diabetes insipidus caused by different molecular mechanisms

Loss-of-function mutations in the V2 vasopressin receptor (AVPR2) gene have been identified as a molecular basis for X-linked nephrogenic diabetes insipidus (NDI). Herein, we describe a novel deletion mutation at nucleotide position 102 (delG102) found in a Russian family resulting in a frameshift and a truncated receptor protein. Furthermore, we analyzed the AVPR2 gene of two other unrelated boys with NDI from our patient clientele. These patients showed previously described mutations (R137H, R181C). In-depth characterization of the three mutant AVPR2s by a combination of functional and immunological techniques permitted further insight into molecular mechanisms leading to receptor dysfunction. Premature truncation of the AVPR2 (delG102) led to a drastically reduced receptor protein expression in transfected COS-7 cells and, as expected, precluded specific AVPR2 functions. As indicated by different ELISA and binding studies, the R137H mutant was almost completely retained in the cell interior. In contrast to previous studies, the few mutant receptors in the plasma membrane displayed a low (2.3-fold above basal) but significant ability to stimulate the G_s/adenylyl cyclase system. In contrast to the latter mutation, the R181C mutant is properly delivered to the cell surface but the mutation interferes with high affinity vasopressin binding. Impaired ligand binding is reflected in an about 100-fold shift of the concentration-response curve toward higher vasopressin concentrations with only slightly reduced agonist potency. Hum Mutat 12:196–205, 1998. © 1998 Wiley-Liss, Inc.     

Two novel mutations in the aquaporin 2 gene in a girl with congenital nephrogenic diabetes insipidus

Congenital nephrogenic diabetes insipidus (CNDI) is a rare inherited disorder characterized by insensitivity of the kidney to the antidiuretic effect of vasopressin. There are three inheritance patterns of CNDI: the X-linked recessive form associated with vasopressin V2 receptor gene mutations, and the autosomal recessive and dominant forms associated with aquaporin-2 gene (AQP2) mutations. The evaluation for polyuria and polydipsia in a one-month-old Korean girl revealed no response to vasopressin and confirmed the diagnosis of CNDI. Because the child was female without family history of CNDI, her disease was thought to be an autosomal recessive form. We analyzed the AQP2 gene and detected a compound heterozygous missense point mutation: 70Ala (GCC) to Asp (GAC) in exon 1 inherited from her father and 187Arg (CGC) to His (CAC) in exon 3 inherited from her mother. The first mutation is located within the first NPA motif of the AQP2 molecule and the second one right after the second NPA motif. This is the first report to characterize AQP2 mutations in Korean patients with autosomal recessive CNDI, and expands the spectrum of AQP2 mutations by reporting two novel mutation, 70Ala (GCC) to Asp (GAC) and 187Arg (CGC) to His (CAC).     

Congenital nephrogenic diabetes insipidus presented with bilateral hydronephrosis: genetic analysis of V2R gene mutations

Most cases of hydronephrosis are caused by urinary tract obstruction. However, excessive polyuric syndrome rarely gives rise to non-obstructive hydronephrosis, megaureter, and a distended bladder. The authors report here on two cases of congenital nephrogenic diabetes insipidus (NDI) with severe bilateral hydronephrosis and megaureter. It is Interesting that the patients were symptomless except for their polyuria, and they both presented with bilateral hydronephrosis. Fluid deprivation testing revealed the presence of AVP resistant NDI. Gene analysis for these patients showed the AVP receptor 2 (V2R) missense mutations (Q225X and S126F), which have previously been reported on in other studies. We made the diagnosis of NDI by using a physiologic test, and we confirmed it by mutation analysis of the V2R gene.     

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.     

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.     

Cognitive and psychosocial functioning of patients with congenital nephrogenic diabetes insipidus

Mental retardation (MR) is generally considered one of the main complications of congenital nephrogenic diabetes insipidus (NDI). However, psychometric studies of NDI patients are scarce and outdated. In the present study, 17 male NDI patients underwent psychological evaluation. Total intelligence quotient of 14 patients was within (n = 13) or above (n = 1) the normal range, 1 patient had an intelligence score between −1 and −2 standard deviations (S.D.) and 2 young patients had a general cognitive index more than 2 S.D. below the norm. Attention deficit hyperactivity disorder criteria were met by 8 out of 17 patients and scores on short-term memory were low in 7 out of 10. No relation between test performances and age at diagnosis or hypernatremia could be found, with the exception of a negative correlation between age at start of therapy and verbal IQ in one age group. Although several explanations for an association between MR and NDI can be postulated, it seems that the current prevalence of MR among patients with this disease is considerably lower than suggested in literature. © 1995 Wiley-Liss, Inc.     

Hemodynamic and coagulation responses to 1-desamino[8-D-arginine] vasopressin in patients with congenital nephrogenic diabetes insipidus

The antidiuretic hormone arginine vasopressin interacts with two types of receptors: V1, which mediates the effects of vasopressin on vascular smooth muscle, and V2, which mediates the antidiuretic effects on renal tubules. Resistance of the renal tubules to arginine vasopressin and to the antidiuretic V2-specific agonist 1-desamino[8-D-arginine] vasopressin (dDAVP) occurs in congenital nephrogenic diabetes insipidus, a rare X-linked disease, although the V1-receptor responses remain intact. The extrarenal actions of dDAVP in normal persons are a decrease in blood pressure, an increase in plasma renin activity, and stimulation of the release of factor VIIIc and von Willebrand factor. We measured the response of mean arterial pressure, pulse rate, plasma renin activity, factor VIIIc, and von Willebrand factor to an infusion of dDAVP (0.3 microgram per kilogram of body weight) in seven male patients with congenital nephrogenic diabetes insipidus, six obligatory carriers of the gene for nephrogenic diabetes insipidus, five patients with central diabetes insipidus, and four normal subjects. In the normal subjects and the patients with central diabetes insipidus, dDAVP decreased mean arterial pressure (by 10 to 15 percent) and increased pulse rate (by 20 to 25 percent), renin activity (by 65 percent), and the release of coagulation factors (twofold to threefold) (all changes were significant, P less than 0.01). None of these changes were observed in the patients with congenital nephrogenic diabetes insipidus, and minimal responses were observed in the obligatory carriers. These results confirm the existence of extrarenal vasopressin V2-like receptors, which may be defective in patients with congenital nephrogenic diabetes insipidus.     

A Null mutation in the vasopressin V2 receptor gene (AVPR2) associated with nephrogenic diabetes insipidus in the Hopewell kindred

Congenital nephrogenic diabetes insipidus (DIR) Is a rare X-linkedhereditary disorder in which the renal collecting duct Is unresponsiveto arginine vasopressin; thus, the urine is consistently hypotonicto plasma. Recently, the association between the V2 receptorgene (AVPR2) and DIR has been proven. We have determined thegene sequence of four family members, from three generations,of a large North American family with CNDI who were originallypart of the study used to formulate the Hopewell hypothesis.It had been proposed that a single DIR gene defect was Introducedto North America by a member of an Ulster Scot kindred arrivingon the ship Hopewell In 1761. DNA sequencing of the AVPR2 hasIdentified a single base transversion from G     

p.R254Q mutation in the aquaporin‐2 water channel causing dominant nephrogenic diabetes insipidus is due to a lack of arginine vasopressin‐induced phosphorylation

Vasopressin regulates human water homeostasis by re‐distributing homotetrameric aquaporin‐2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells, a process in which phosphorylation of AQP2 at S256 by cAMP‐dependent protein kinase A (PKA) is thought to be essential. Dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by AQP2 gene mutations. Here, we investigated a reported patient case of dominant NDI caused by a novel p.R254Q mutation. Expressed in oocytes, AQP2‐p.R254Q appeared to be a functional water channel, but was impaired in its transport to the cell surface to the same degree as AQP2‐p.S256A, which mimics non‐phosphorylated AQP2. In polarized MDCK cells, AQP2‐p.R254Q was retained and was distributed similarly to that of unstimulated wt‐AQP2 or AQP2‐p.S256A. Upon co‐expression, AQP2‐p.R254Q interacted with, and retained wt‐AQP2 in intracellular vesicles. In contrast to wild‐type AQP2, forskolin did not increase AQP2‐p.R254Q phosphorylation at S256 or its translocation to the apical membrane. Mimicking constitutive phosphorylation in AQP2‐p.R254Q with the p.S256D mutation, however, rescued its apical membrane expression. These date indicate that a lack of S256 phosphorylation is the sole cause of dominant NDI here, and thereby, p.R254Q is a loss of function instead of a gain of function mutation in dominant NDI. © 2009 Wiley‐Liss, Inc.     

A novel mechanism in recessive nephrogenic diabetes insipidus: wild-type aquaporin-2 rescues the apical membrane expression of intracellularly retained AQP2-P262L

Vasopressin regulates water homeostasis through insertion of homotetrameric aquaporin-2 (AQP2) water channels in the apical plasma membrane of renal cells. AQP2 mutations cause recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Until now, all AQP2 mutants in recessive NDI were shown to be misfolded, retained in the endoplasmic reticulum (ER) and unable to interact with wild-type (wt)-AQP2, whereas AQP2 mutants in dominant NDI are properly folded and interact with wt-AQP2, but, due to the mutation, cause missorting of the wt-AQP2/mutant complex. Here, patients of two families with recessive NDI appeared compound heterozygotes for AQP2-A190T or AQP2-R187C mutants, together with AQP2-P262L. As mutations in the AQP2 C-tail, where P262 resides, usually cause dominant NDI, the underlying cell-biological mechanism was investigated. Upon expression in oocytes, AQP2-P262L was a properly folded and functional aquaporin in contrast to the classical mutants, AQP2-R187C and AQP2-A190T. Expressed in polarized cells, AQP2-P262L was retained in intracellular vesicles and did not localize to the ER. Upon co-expression, however, AQP2-P262L interacted with wt-AQP2, but not with AQP2-R187C, resulting in a rescued apical membrane expression of AQP2-P262L. In conclusion, our study reveals a novel cellular phenotype in recessive NDI in that AQP2-P262L acts as a mutant in dominant NDI, except for that its missorting is overruled by apical sorting of wt-AQP2. Also, it demonstrates for the first time that the recessive inheritance of a disease involving a channel can be due to two cell-biological mechanisms.     

Contiguous gene deletion involving L1CAM and AVPR2 causes X-linked hydrocephalus with nephrogenic diabetes insipidus

X-linked hydrocephalus with aqueductal stenosis (HSAS) is caused by mutation or deletion of the L1 cell adhesion molecule gene (L1CAM) at Xq28. Central diabetes insipidus (CDI) can arise as a consequence of resultant hypothalamic dysfunction from hydrocephalus and must be distinguished from nephrogenic diabetes insipidus (NDI) by exogenous vasopressin response. Causes of NDI are heterogeneous and include mutation or deletion of the arginine vasopressin receptor 2 gene (AVPR2), which is located approximately 29 kb telomeric to L1CAM. We identified a patient with both HSAS and NDI where DNA sequencing failure suggested the possibility of a contiguous gene deletion. A 32.7 kb deletion mapping from L1CAM intron1 to AVPR2 exon2 was confirmed. A 90 bp junctional insertion fragment sharing short direct repeat homology with flanking sequences was identified. To our knowledge this is the first reported case of an Xq28 microdeletion involving both L1CAM and AVPR2, defining a new contiguous gene syndrome comprised of HSAS and NDI. Contiguous gene deletion should be considered as a mechanism for all patients presenting with hydrocephalus and NDI.     

Immunohistochemical characterization of the intracellular pool of water channel aquaporin-2 in the rat kidney

Aquaporin-2 (AQP2) is a member of water channel proteins expressed in the kidney collecting duct cells, where it is stored in the intracellular compartment. Upon stimulation of antidiuretic hormone (ADH), AQP2 is recruited to the plasma membrane, and plays a critical role in urine concentration. We immunohistochemically characterized the intracellular compartment harboring AQP2 in the rat kidney using antibodies to the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, lysosome, and endosome. Aquaporin-2 did not colocalize with calnexin, TGN38, Golgi 58K, cathepsin D or lgp-110. Small portions of AQP2-bearing vesicles were positive for early endosome antigen 1. These localization patterns were basically the same in water-loaded and ADH-treated animals. These results indicate that AQP2-bearing vesicles constitute a unique intracellular compartment distinct from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network and lysosome. Partial colocalization of AQP2 with early endosomes suggests that the endosomal system might be involved in the trafficking of AQP2.     

C112R, W323S, N317K mutations in the vasopressin V2 receptor gene in patients with nephrogenic diabetes insipidus. Mutations in brief no. 165. Online

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 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 further evidence that X-linked NDI results from defects in the V2 receptor gene.     

Identification and characterization of 15 novel GALC gene mutations causing Krabbe disease

The characterization of the underlying GALC gene lesions was performed in 30 unrelated patients affected by Krabbe disease, an autosomal recessive leukodystrophy caused by the deficiency of lysosomal enzyme galactocerebrosidase. The GALC mutational spectrum comprised 33 distinct mutant (including 15 previously unreported) alleles. With the exception of 4 novel missense mutations that replaced evolutionarily highly conserved residues (p.P318R, p.G323R, p.I384T, p.Y490N), most of the newly described lesions altered mRNA processing. These included 7 frameshift mutations (c.61delG, c.408delA, c.521delA, c.1171_1175delCATTCinsA, c.1405_1407delCTCinsT, c.302_308dupAAATAGG, c.1819_1826dupGTTACAGG), 3 nonsense mutations (p.R69X, p.K88X, p.R127X) one of which (p.K88X) mediated the skipping of exon 2, and a splicing mutation (c.1489+1G>A) which induced the partial skipping of exon 13. In addition, 6 previously unreported GALC polymorphisms were identified. The functional significance of the novel GALC missense mutations and polymorphisms was investigated using the MutPred analysis tool. This study, reporting one of the largest genotype-phenotype analyses of the GALC gene so far performed in a European Krabbe disease cohort, revealed that the Italian GALC mutational profile differs significantly from other populations of European origin. This is due in part to a GALC missense substitution (p.G553R) that occurs at high frequency on a common founder haplotype background in patients originating from the Naples region.