Mutations in Na‐K‐2Cl co‐transporter, NKCC2, lead to type I Bartter syndrome (BS1), a life‐threatening kidney disease. Yet, our knowledge of the molecular regulation of NKCC2 mutants remains poor. ...Here, we aimed to identify the molecular pathogenic mechanisms of one novel and three previously reported missense NKCC2 mutations. Co‐immunolocalization studies revealed that all NKCC2 variants are not functional because they are not expressed at the cell surface due to retention in the endoplasmic reticulum (ER). Cycloheximide chase assays together with treatment by protein degradation and mannose trimming inhibitors demonstrated that the defect in NKCC2 maturation arises from ER retention and associated degradation (ERAD). Small interfering RNA (siRNA) knock‐down experiments revealed that the ER lectin OS9 is involved in the ERAD of NKCC2 mutants. 4‐phenyl butyric acid (4‐PBA) treatment mimicked OS9 knock‐down effect on NKCC2 mutants by stabilizing their immature forms. Importantly, out of the four studied mutants, only one showed an increased protein maturation upon treatment with glycerol. In summary, our study reveals that BS1 is among diseases linked to the ERAD pathway. Moreover, our data open the possibility that maturation of some ER retained NKCC2 variants is correctable by chemical chaperones offering, therefore, promising avenues in elucidating the molecular pathways governing the ERAD of NKCC2 folding mutants.
All NKCC2 mutants, related to Bartter syndrome type 1, are not functional because they are not expressed at the cell surface due to retention in the endoplasmic reticulum. The ER associated‐degradation (ERAD) of NKCC2 mutants involves the ER lectin OS9. 4‐PBA treatment inhibits the ERAD of all NKCC2 variants. The maturation of some ER retained NKCC2 mutants is rescuable, at least partially, by chemical chaperones opening therefore the possibility that Bartter syndrome type 1 can be potentially treated with chemical and/or pharmacological chaperones.
Mutations in the CLCN5 gene encoding the 2Cl−/1H+ exchanger ClC‐5 are associated with Dent disease 1, an inherited renal disorder characterized by low‐molecular‐weight (LMW) proteinuria and ...hypercalciuria. In the kidney, ClC‐5 is mostly localized in proximal tubule cells, where it is thought to play a key role in the endocytosis of LMW proteins. Here, we investigated the consequences of eight previously reported pathogenic missense mutations of ClC‐5 surrounding the “proton glutamate” that serves as a crucial H+‐binding site for the exchanger. A complete loss of function was observed for a group of mutants that were either retained in the endoplasmic reticulum of HEK293T cells or unstainable at plasma membrane due to proteasomal degradation. In contrast, the currents measured for the second group of mutations in Xenopus laevis oocytes were reduced. Molecular dynamics simulations performed on a ClC‐5 homology model demonstrated that such mutations might alter ClC‐5 protonation by interfering with the water pathway. Analysis of clinical data from patients harboring these mutations demonstrated no phenotype/genotype correlation. This study reveals that mutations clustered in a crucial region of ClC‐5 have diverse molecular consequences in patients with Dent disease 1, ranging from altered expression to defects in transport.
Dent disease type 1 is a renal tubular disorder characterized by low‐molecular‐weight proteinuria, hypercalciuria with nephrocalcinosis or nephrolithiasis, and progressive renal failure. It is associated with mutations in the gene encoding the 2Cl‐/H+ exchanger ClC‐5. Here, we have investigated the consequences of eight previously reported pathogenic missense mutations of ClC‐5 surrounding the ‘proton glutamate’ (E268), a crucial H+−binding site for the exchanger. We have demonstrated that these mutations have diverse consequences, ranging from altered expression to defects in ion transport. In this later case, we demonstrated that ClC−5 protonation might be altered by disruption of the water pathway of the transporter.
Introduction
Prenatal diagnosis of bone and mineralization anomalies is associated with a wide range of etiologies and prognoses. The improvement of antenatal ultrasound combined with the development ...of molecular diagnosis in genetics has transformed antenatal medicine into a challenging discipline. Of the various known causes of bone abnormalities and hypomineralization, calcium and phosphate metabolism disorders are exceptional. An accurate diagnosis is crucial for providing appropriate genetic counseling and medical follow‐up after birth.
Case
We report on three siblings with severe bone abnormalities diagnosed during the second trimester ultrasound of pregnancy. Postnatal follow‐up showed transitory hyperparathyroidism, with hypercalcemia and hypocalciuria.
Methods
Sanger sequencing performed after birth in the three newborns revealed a monoallelic pathogenic variant in the CASR gene, encoding the calcium sensing receptor, confirming the diagnosis of familial hypocalciuric hypercalcemia, paternally inherited. Postnatal evolution was favorable after treatment with a calcimimetic agent.
Conclusions
Previously, prenatal bone abnormalities caused by familial hypocalciuric hypercalcemia had only been described in one patient. This entity should be considered as differential diagnosis of bones abnormalities. Knowing about this unusual etiology is important to guide the diagnosis, the prenatal counseling and to improve medical management.
Highlights
What is already know?
Familial hypocalciuric hypercalcemia type 1 (FHH1) is a genetic disorder, caused mainly by heterozygous mutations in the CASR gene. It associates hypercalcemia, normal PTH value, and normo‐ or hypocalciuria. Some children may present with bone abnormalities and early hyperparathyroidism, resolving gradually.
What does this study add?
This article presents three cases of prenatal bone abnormalities due to postnatally genetically confirmed FHH that resolved within the first years of life. This condition should be considered as a differential diagnosis in antenatal bone abnormalities.
Objective
Familial hypocalciuric hypercalcaemia type 1 (FHH1), related to heterozygous loss‐of‐function mutations of the calcium‐sensing receptor gene, is the main differential diagnosis for primary ...hyperparathyroidism. The aim of our study was to describe clinical characteristics of adult patients living in France with a genetically confirmed FHH1.
Design and patients
This observational, retrospective, multicentre study included 77 adults, followed up in 32 clinical departments in France, with a genetic FHH1 diagnosis between 2001 and 2012.
Results
Hypercalcaemia was diagnosed at a median age of 53 years IQR: 38‐61. The diagnosis was made after clinical manifestations, routine analysis or familial screening in 56, 34 and 10% of cases, respectively, (n = 58; data not available for 19 patients). Chondrocalcinosis was present in 11/51 patients (22%), bone fractures in 8/56 (14%) and renal colic in 6/55 (11%). The median serum calcium was 2.74 mmol/L IQR: 2.63‐2.86 mmol/L, the median plasma parathyroid hormone level was 4.9 pmol/L 3.1‐7.1, and the median 24‐hour urinary calcium excretion was 2.8 mmol/24 hours IQR: 1.9‐4.0. Osteoporosis (dual X‐ray absorptiometry) or kidney stones (renal ultrasonography) were found in 6/38 patients (16%) and 9/32 patients (28%), respectively. Fourteen patients (18%) underwent parathyroid surgery; parathyroid adenoma was found in three patients (21%) and parathyroid hyperplasia in nine patients (64%). No correlation between genotype and phenotype was established.
Conclusion
This large cohort study demonstrates that FHH1 clinical characteristics can be atypical in 33 patients (43%). Clinicians should be aware of this rare differential diagnosis in order to adopt an appropriate treatment strategy.
Dent disease is an X‐linked recessive renal tubular disorder characterized by low‐molecular‐weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis, and progressive renal failure. ...Inactivating mutations of CLCN5, the gene encoding the 2Cl−/H+ exchanger ClC‐5, have been reported in patients with Dent disease 1. In vivo studies in mice harboring an artificial mutation in the “gating glutamate” of ClC‐5 (c.632A > C, p.Glu211Ala) and mathematical modeling suggest that endosomal chloride concentration could be an important parameter in endocytosis, rather than acidification as earlier hypothesized. Here, we described a novel pathogenic mutation affecting the “gating glutamate” of ClC‐5 (c.632A>G, p.Glu211Gly) and investigated its molecular consequences. In HEK293T cells, the p.Glu211Gly ClC‐5 mutant displayed unaltered N‐glycosylation and normal plasma membrane and early endosomes localizations. In Xenopus laevis oocytes and HEK293T cells, we found that contrasting with wild‐type ClC‐5, the mutation abolished the outward rectification, the sensitivity to extracellular H+ and converted ClC‐5 into a Cl− channel. Investigation of endosomal acidification in HEK293T cells using the pH‐sensitive pHluorin2 probe showed that the luminal pH of cells expressing a wild‐type or p.Glu211Gly ClC‐5 was not significantly different. Our study further confirms that impaired acidification of endosomes is not the only parameter leading to defective endocytosis in Dent disease 1.
Dent disease type 1, a renal tubular disorder characterized by low‐molecular‐weight proteinuria, hypercalciuria, nephrocalcinosis, and progressive renal failure is associated with mutations in the gene encoding the 2Cl−/H+ exchanger ClC‐5. This transporter plays a crucial role in receptor‐mediated endocytosis by permitting acidification of the early endosomes of the proximal tubule. We have demonstrated that a novel mutation affecting the “gating glutamate” converts ClC‐5 into a Cl− channel without impairing acidification of endosomes.
Gitelman syndrome (GS) is a rare, salt-losing tubulopathy characterized by hypokalemic metabolic alkalosis with hypomagnesemia and hypocalciuria. The disease is recessively inherited, caused by ...inactivating mutations in the SLC12A3 gene that encodes the thiazide-sensitive sodium-chloride cotransporter (NCC). GS is usually detected during adolescence or adulthood, either fortuitously or in association with mild or nonspecific symptoms or both. The disease is characterized by high phenotypic variability and a significant reduction in the quality of life, and it may be associated with severe manifestations. GS is usually managed by a liberal salt intake together with oral magnesium and potassium supplements. A general problem in rare diseases is the lack of high quality evidence to inform diagnosis, prognosis, and management. We report here on the current state of knowledge related to the diagnostic evaluation, follow-up, management, and treatment of GS; identify knowledge gaps; and propose a research agenda to substantiate a number of issues related to GS. This expert consensus statement aims to establish an initial framework to enable clinical auditing and thus improve quality control of care.
Bartter syndrome is a rare inherited salt-losing renal tubular disorder characterized by secondary hyperaldosteronism with hypokalemic and hypochloremic metabolic alkalosis and low to normal blood ...pressure. The primary pathogenic mechanism is defective salt reabsorption predominantly in the thick ascending limb of the loop of Henle. There is significant variability in the clinical expression of the disease, which is genetically heterogenous with 5 different genes described to date. Despite considerable phenotypic overlap, correlations of specific clinical characteristics with the underlying molecular defects have been demonstrated, generating gene-specific phenotypes. As with many other rare disease conditions, there is a paucity of clinical studies that could guide diagnosis and therapeutic interventions. In this expert consensus document, the authors have summarized the currently available knowledge and propose clinical indicators to assess and improve quality of care.
The thick ascending limb plays a central role in human kidney physiology, participating in sodium reabsorption, urine concentrating mechanisms, calcium and magnesium homeostasis, bicarbonate and ...ammonium homeostasis, and uromodulin synthesis. This review aims to illustrate the importance of these roles from a pathophysiological point of view by describing the interactions of the key proteins of this segment and by discussing how recently identified and long-known hereditary diseases affect this segment. The descriptions of two recently described salt-losing tubulopathies, transient antenatal Bartter syndrome and HELIX syndrome, which are caused by mutations in
MAGED2
and
CLDN10
genes, respectively, highlight the role of new players in the modulation of sodium reabsorption the thick ascending limb.
Gitelman syndrome is an autosomal recessive tubulopathy characterized by hypokalemia, hypomagnesemia, metabolic alkalosis and hypocalciuria. The majority of patients do not present with symptoms ...until late childhood or adulthood, and the symptoms are generally mild. We report here the first case of Gitelman syndrome presenting with the biological features of Fanconi syndrome and an early polyuria since the neonatal period. We discuss in this article the atypical electrolytes losses found in our patient, as well as the possible mechanisms of severe polyuria.
A 6-year-old Caucasian girl was admitted via the Emergency department for vomiting, and initial laboratory investigations found hyponatremia, hypokalemia, metabolic acidosis with normal anion gap, hypophosphatemia, and hypouricemia. Urinalysis revealed Na, K, Ph and uric acid losses. Thus, the initial biological profile was in favor of a proximal tubular defect. However, etiological investigations were inconclusive and the patient was discharged with potassium chloride and phosphorus supplementation. Three weeks later, further laboratory analysis indicated persistent hypokalemia, a metabolic alkalosis, hypomagnesemia, and hypocalciuria. We therefore sequenced the SLC12A3 gene and found a compound heterozygosity for 2 known missense mutations.
Gitelman syndrome can have varying and sometimes atypical presentations, and should be suspected in case of hypokalemic tubular disorders that do not belong to any obvious syndromic entity. In this case, the proximal tubular dysfunction could be secondary to the severe hypokalemia. This report emphasizes the need for clinicians to repeat laboratory tests in undiagnosed tubular disorders, especially not during decompensation episodes.
ABSTRACT
Dent disease is a rare X‐linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and ...variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC‐5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5‐phosphatase OCRL‐1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in‐frame mutations described were mapped onto a three‐dimensional homology model of the ClC‐5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.
We review previously reported mutations in the CLCN5 gene (Dent disease 1) and describe phenotype, novel (n=42) and recurrent mutations (n=24) in a large cohort of 117 patients belonging to 90 families. The novel missense and in‐frame mutations were mapped onto a three‐dimensional homology model of the ClC‐5 protein: this analysis suggests that these mutations affect the dimerization process, helix stability or transport. The phenotype of our cohort patients supports and extends the phenotype previously reported in smaller studies.
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