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Contains fulltext : 244896.pdf (Publisher’s version ) (Closed access) BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function. 01 november 2021

Background Nephronophthisis (NPH) is the most prevalent genetic cause for ESRD in children. However, little is known about the prevalence of NPH in adult-onset ESRD. Homozygous full gene deletions of the NPHP1 gene encoding nephrocystin-1 are a prominent cause of NPH. We determined the prevalence of NPH in adults by assessing homozygous NPHP1 full gene deletions in adult-onset ESRD.Methods Adult renal transplant recipients from five cohorts of the International Genetics and Translational Research in Transplantation Network (iGeneTRAiN) underwent single-nucleotide polymorphism genotyping. After quality control, we determined autosomal copy number variants (such as deletions) on the basis of median log2 ratios and B-allele frequency patterns. The findings were independently validated in one cohort. Patients were included in the analysis if they had adult-onset ESRD, defined as start of RRT at >= 18 years old.Results We included 5606 patients with adult-onset ESRD; 26 (0.5%) showed homozygous NPHP1 deletions. No donor controls showed homozygosity for this deletion. Median age at ESRD onset was 30 (range, 18-61) years old for patients with NPH, with 54% of patients age >= 30 years old. Notably, only three (12%) patients were phenotypically classified as having NPH, whereas most patients were defined as having CKD with unknown etiology (n=11; 42%).Conclusions Considering that other mutation types in NPHP1 or mutations in other NPH-causing genes were not analyzed, NPH is a relatively frequent monogenic cause of adult-onset ESRD. Because 88% of patients had not been clinically diagnosed with NPH, wider application of genetic testing in adult-onset ESRD may be warranted.

Contains fulltext : 53685.pdf (Publisher’s version ) (Open Access) Primary nocturnal enuresis (PNE) is a common problem in childhood and adolescence. Although various treatments are highly effective, a common underlying hypothesis on the pathogenesis is lacking. The success of desmopressin, a synthetic analogue of the antidiuretic hormone vasopressin, has been attributed to increased renal water reabsorption that is mediated by activation of the renal vasopressin V2 receptor (V2R). However, this effect does not explain other symptoms of PNE, such as the failure to arouse upon bladder distension. This study identified a family in which one child displayed PNE and coexisting nephrogenic diabetes insipidus, as a result of a novel nonsense mutation in the V2R gene (C358X). Cell-biologic investigations revealed that V2R-C358X is retained in the endoplasmic reticulum and is unstable, which explains his nephrogenic diabetes insipidus. Consistently, extrarenal V2R-mediated responses were absent in the patient who was treated with desmopressin. Administration of desmopressin, however, changed his PNE into nocturia, because he now still voided unchanged high urinary volumes at night but woke up and went to the bathroom. Withdrawal of desmopressin was accompanied by bedwetting, whereas reintroduction again relieved the symptoms. Therefore, these data indicate that neither a functioning renal concentration system nor a functional V2R is needed for the therapeutic benefit of desmopressin in PNE. Rather, it suggests that another vasopressin receptor and other organ(s) is the target for desmopressin to relieve PNE.

Item does not contain fulltext Bartter syndrome (BS) is a heterogeneous renal tubular disorder affecting Na-K-Cl reabsorption in the thick ascending limb of Henle's loop. BS type I patients typically present with profound hypokalemia and metabolic alkalosis. The main goal of the present study was to elucidate the functional implications of six homozygous mutations (G193R, A267S, G319R, A508T, del526N, and Y998X) in the bumetanide-sensitive Na-K-2Cl cotransporter (hNKCC2) identified in patients diagnosed with BS type I. To this end, capped RNA (cRNA) of FLAG-tagged hNKCC2 and the corresponding mutants was injected in Xenopus laevis oocytes and transporter activity was measured after 72 h by means of a bumetanide-sensitive (22)Na(+) uptake assay at 30 degrees C. Injection of 25 ng of hNKCC2 cRNA resulted in bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.5 nmol/oocyte per 30 min. Injection of 25 ng of mutant cRNA yielded no significant bumetanide-sensitive (22)Na(+) uptake. Expression of wild-type and mutant transporters was confirmed by immunoblotting, showing significantly less mutant protein compared with wild-type at the same cRNA injection levels. However, when the wild-type cRNA injection level was reduced to obtain a protein expression level equal to that of the mutants, the wild-type still exhibited a significant bumetanide-sensitive (22)Na(+) uptake. Immunocytochemical analysis showed immunopositive staining of hNKCC2 at the plasma membrane for wild-type and all studied mutants. In conclusion, mutations in hNKCC2 identified in type I BS patients, when expressed in Xenopus oocytes, result in a low expression of normally routed but functionally impaired transporters. These results are in line with the hypothesis that the mutations in hNKCC2 are the underlying cause of the clinical abnormalities seen in patients with type I BS.

Mutations in the Aquaporin-2 gene, which encodes a renal water channel, have been shown to cause autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Most AQP2 missense mutants in recessive NDI are retained in the endoplasmic reticulum (ER), but AQP2-T125M and AQP2-G175R were reported to be nonfunctional channels unimpaired in their routing to the plasma membrane. In five families, seven novel AQP2 gene mutations were identified and their cell-biologic basis for causing recessive NDI was analyzed. The patients in four families were homozygous for mutations, encoding AQP2-L28P, AQP2-A47V, AQP2-V71M, or AQP2-P185A. Expression in oocytes revealed that all these mutants, and also AQP2-T125M and AQP2-G175R, conferred a reduced water permeability compared with wt-AQP2, which was due to ER retardation. The patient in the fifth family had a G>A nucleotide substitution in the splice donor site of one allele that results in an out-of-frame protein. The other allele has a nucleotide deletion (c652delC) and a missense mutation (V194I). The routing and function of AQP2-V194I in oocytes was not different from wt-AQP2; it was therefore concluded that c652delC, which leads to an out-of-frame protein, is the NDI-causing mutation of the second allele. This study indicates that misfolding and ER retention is the main, and possibly only, cell-biologic basis for recessive NDI caused by missense AQP2 proteins. In addition, the reduced single channel water permeability of AQP2-A47V (40%) and AQP2-T125M (25%) might become of therapeutic value when chemical chaperones can be found that restore their routing to the plasma membrane.

Nail-patella syndrome is an autosomal dominant disorder characterized by dyplasia of finger nails, skeletal anomalies, and, frequently, renal disease. It has recently been shown that this disorder is caused by putative loss-of-function mutations in a transcription factor (LMX1B) belonging to the LIM-homeodomain family, members of which are known to be important for pattern formation during development. A cohort of eight Dutch NPS families were screened for mutations in the LMX1B gene; seven different mutations, including one novel variant, were identified. Three of the mutations are very likely to result in truncated LMX1B proteins, three are predicted to influence sequence-specific DNA binding, and one is presumed to prevent the formation of a stable protein by abolishing the Zn(II) binding site of the protein. Although there was a remarkable high incidence of renal disease in one of the families, the nephropathy was not seen in all affected family members and the severity of renal impairment varied significantly among the patients. This indicates that the incidence and severity of nephropathy within this family cannot be attributed to the LMX1B genotype. In addition, evidence of a correlation between other characteristics of the NPS phenotype and specific mutations has not been found.

Inherited hypokalemic renal tubulopathies are differentiated into at least three clinical subtypes: (1) the Gitelman variant of Bartter syndrome (GS); (2) hyperprostaglandin E syndrome, the antenatal variant of Bartter syndrome (HPS/aBS); and (3) the classic Bartter syndrome (cBS). Hypokalemic metabolic alkalosis and renal salt wasting are the common characteristics of all three subtypes. Hypocalciuria and hypomagnesemia are specific clinical features of Gitelman syndrome, while HPS/aBS is a life-threatening disorder of the newborn with polyhydramnios, premature delivery, hyposthenuria, and nephrocalcinosis. The Gitelman variant is uniformly caused by mutations in the gene for the thiazide-sensitive NaCl-cotransporter NCCT (SLC12A3) of the distal tubule, while HPS/aBS is caused by mutations in the gene for either the furosemide-sensitive NaK-2Cl-cotransporter NKCC2 (SLC12A1) or the inwardly rectifying potassium channel ROMK (KCNJ1). Recently, mutations in a basolateral chloride channel CLC-Kb (CLCNKB) have been described in a subset of patients with a Bartter-like phenotype typically lacking nephrocalcinosis. In this study, the screening for CLCNKB mutations showed 20 different mutations in the affected children from 30 families. The clinical characterization revealed a highly variable phenotype ranging from episodes of severe volume depletion and hypokalemia during the neonatal period to almost asymptomatic patients diagnosed during adolescence. This study adds 16 novel mutations to the nine already described, providing further evidence that mutations in the gene for the basolateral chloride channel CLC-Kb are the molecular basis of classic Bartter syndrome. Interestingly, the phenotype elicited by CLCNKB mutations occasionally includes HPS/aBS, as well as a Gitelman-like phenotype.

Nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine in response to vasopressin. The autosomal recessive form of NDI is caused by mutations in the AQP2 gene, encoding the vasopressin-regulated water channel of the kidney collecting duct. This report presents three new mutations in the AQP2 gene that cause NDI, resulting in A147T-, T126M-, or N68S-substituted AQP2 proteins. Expression of the A147T and T126M mutant AQP2 proteins in Xenopus oocytes revealed a relatively small, but significant increase in water permeability, whereas the water permeability of N68S expressing oocytes was not increased. cRNA encoding missense and wild-type AQP2 were equally stable in oocytes. Immunoblots of oocyte lysates showed that only the A147T mutant protein was less stable than wild-type AQP2. The mutant AQP2 proteins showed, in addition to the wild-type 29-kd band, an endoplasmic reticulum-retarded form of AQP2 of approximately 32 kd. Immunoblotting and immunocytochemistry demonstrated only intense labeling of the plasma membranes of oocytes expressing wild-type AQP2. In summary, two mutant AQP2 proteins encoded in NDI are functional water channels. Therefore, the major cause underlying autosomal recessive NDI is the misrouting of AQP2 mutant proteins.