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Unusual Clinical Presentation and Possible Rescue of a Novel Claudin-16 Mutation

Department of Pediatric Nephrology (D.M., J.T.), Charité Children’s Hospital and Center for Cardiovascular Research, 12200 Berlin, Germany; Epithelial Cell Biology Laboratory (P.J.K., W.H.), Institute of Molecular and Cell Biology, Singapore 138673; Great Ormond Street Hospital for Children National Health Service Trust (D.B., W.v.H.), London WC1N 3JH, United Kingdom; Max-Delbrueck-Centre for Molecular Medicine (I.C.M.), 12200 Berlin, Germany; and Institute of Child Health (M.J.D.), London WC1N 3JH, United Kingdom

Address all correspondence and requests for reprints to: Dr. Walter Hunziker, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673. E-mail: hunziker{at}imcb.a-star.edu.sg.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion Note Added in Proof References

 
Context: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is caused by a dysfunction of Claudin-16 (CLDN16) and characterized by renal wasting of Mg²⁺ and Ca²⁺.

Objective: The objectives of this study were to study the clinical parameters in suspected FHHNC patients, identify mutations in the CLDN16 gene, and analyze molecular defects associated with the mutant protein.

Design, Setting, and Participants: CLDN16 genes from two siblings diagnosed with FHHNC were sequenced. Expression and characterization of the mutant protein in renal MDCK cells were studied.

Outcome Measures: Standard urine and serum parameters to diagnose FHHNC were determined. Mutations in the CLDN16 gene were identified. The subcellular distribution of the mutant protein was analyzed by immunofluorescence microscopy.

Results: Urine and blood analysis showed signs typical for FHHNC. One patient, in addition, presented with hypocalcemic tetany, a phenomenon so far not described for FHHNC. Both siblings carry a novel mutation in CLDN16, Y207X. The review of medical records showed that hypocalcemia is not uncommon in the early childhood of FHHNC patients. Expressed in MDCK cells, the Y207X mutant is not detected at tight junctions but instead is found in lysosomes and, to a lesser extent, the endoplasmic reticulum. Surface expression can be rescued by inhibiting clathrin-mediated internalization.

Conclusions: We propose that mutations in CLDN16 are considered in childhood hypocalcemia. CLDN16 Y207X is transiently delivered to the plasma membrane but not retained and is rapidly retrieved by internalization. Inhibitors of endocytosis may provide novel therapeutic strategies.

	Introduction

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THE KIDNEY PLAYS a crucial role in maintaining overall Ca²⁺ and Mg²⁺ homeostasis. This role is critically dependent on the ability to reabsorb water and solutes along the renal tubular epithelium after glomerular filtration. This is accomplished by several renal transcellular transport systems combined with paracellular transport mediated by the intercellular tight junction (TJ). TJs consist of distinct transmembrane proteins that are tethered to the actin cytoskeleton via scaffolding proteins (1). Claudins, a superfamily of transmembrane proteins comprising 24 members in eukaryotes (2), are essential components of TJs. CLDN16 is exclusively expressed in the thick ascending loop of Henle, where it participates in Mg²⁺ and Ca²⁺ transport (3). The crucial role of CLDN16 for human Mg²⁺ homeostasis is underlined by the identification of mutations in the CLDN16 gene of patients suffering from severe renal Mg²⁺ and Ca²⁺ wasting (4). The resulting disorder, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) (OMIM 603959), usually leads to end stage renal disease (ESRD) in the second to fourth decades of life. The only known cure is renal transplantation and symptomatic treatment (i.e. Mg²⁺-citrate, Ca²⁺ replacement, or hydrochlorothiazide) may only delay the onset of ESRD (5). The clinical appearance in affected patients is often uniform, although time to ESRD shows, even among siblings, considerable variation. Differential diagnosis of renal Mg²⁺ wasting includes several disorders. Primary hypomagnesemia and secondary hypocalcemia due to mutations in the gene of the Mg²⁺ channel TRPM6 leads, in general, to significantly lower Mg²⁺ levels than those encountered in FHHNC. Mutations in the -subunit of the renal Na⁺-K⁺-ATPase are inherited in a dominant manner. The Barter and Gitelman’s syndrome can be distinguished by a concomitant loss of Na⁺ and K⁺.

Over 20 different mutations in CLDN16 have been identified, and all are located within the extracellular loops or transmembrane domains (6) and associated with a severe phenotype (7). Only the T233R mutant seems to be linked with a mild clinical course (6). Here, we describe a novel CLDN16 mutation in two affected siblings. Unexpectedly, one sibling presented with hypocalcemic tetany. We also provide insight into the molecular defect of this mutant and a possible therapeutic strategy based on endocytosis inhibitors.

	Patients and Methods

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Case report

Two siblings were diagnosed with FHHNC. The parents come from the same village in Pakistan but are not knowingly related. A third sibling was screened but had a normal renal ultrasound and serum chemistries. The first sibling (female) presented at 6 wk of life with hypocalcemic seizures. Calcium and magnesium serum levels were 1.32 and 0.48 mmol/liter, respectively (Table 1), with normal albumin levels. A 25-OH vitamin D level returned at 17 mg/liter (normal, 3–50). Calcium and vitamin D supplements alleviated the seizures, and the supplements were weaned off at 1 yr of age. A renal ultrasound obtained at that time showed signs of early nephrocalcinosis. At 4 yr of age, she was brought to medical attention again because of inconsolable crying at night. The subsequent work-up confirmed bilateral nephrocalcinosis and eventually led to the diagnosis of FHHNC (Table 1). The other sibling (male) presented at the age of 12 with macroscopic hematuria and flank pain and was found to have a right ureteric stone, dense nephrocalcinosis, and additional stones in both kidneys, for which he underwent three lithotripsy procedures over the following year. Blood and urine chemistries confirmed the diagnosis of FHHNC (Table 1). Since establishing their diagnosis, both siblings are on magnesium supplementation, a thiazide diuretic, and treatment with calcitriol. Informed consent for the studies and publication of the results was obtained from the parents under a Research Ethics Committee-approved protocol.

Mutational analysis of CLDN16 and isolation and epitope tagging of a human CLDN16 cDNA have been described (6). The Y207X mutation was introduced by PCR using suitable overlapping primers and verified by sequencing. A polyclonal antibody to ZO-1 (Zymed Laboratories, South San Francisco, CA), a rat and mouse monoclonal antibody to HA (Roche Diagnostics, Mannheim, Germany) and Lamp-2 (AC-17) (8), respectively, and an affinity-purified polyclonal antibody to the first extracellular loop of CLDN16 (amino acids 52–66; antiloop antibody) (9) were used.

Confocal immunofluorescence microscopy

MDCK cells were cultured and transiently transfected (30–40% efficiency, Lipofectamine, Invitrogen, Carlsbad, CA) as described (6, 10, 11) and used for experiments 24 h later. Cells grown on coverslips were processed for immunofluorescence microscopy (6, 10, 11) using antibodies to HA (1:100), calreticulin (1:300), and Lamp-2 (1:20) and suitable fluorescently labeled secondary antibodies (Molecular Probes, Eugene, OR). Cell surface expression and endocytosis of CLDN16 were monitored by incubating live cells with the antiloop antibody for 1 h at 37 C at a dilution that did not stain control cells. The antiloop antibody was then detected with labeled secondary antibodies. Clathrin-mediated internalization was blocked using cytosol acidification (12) or hypertonic media (13) protocols.

	Results

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Sequence analysis of the CLDN16 gene revealed in both affected patients a novel homozygous thymidine to guanidine change at position 620, resulting in the replacement of tyrosine at position 207 with a premature stop codon (Fig. 1A), thus deleting a large portion of the C-terminal cytosolic domain in the encoded protein (Fig. 1B).

View larger version (39K): [in this window] [in a new window]   FIG. 1. Identification and effect of the Y207X mutation in the CLDN16 gene and subcellular localization, transient surface exposure, and rescue of surface expression of CLDN16 Y207X in transiently transfected MDCK cells. A, Sequencing data for the region in the CLDN16 gene of two siblings carrying the Y207X mutation. The chromatograms read from 5' (left) to 3' (right). *, Homozygous mutation leading to FHHNC. B, Predicted topology of CLDN16. Shown is the amino acid sequence in the one-letter code, with the previously described T233R mutation in the cytosolic tail in yellow. The Y207X mutation described in this study is shown in red and leads to the deletion of a large portion of the cytosolic tail of CLDN16. X, Stop codon. C, Subcellular localization, transient surface exposure, and rescue of surface expression of CLDN16 Y207X in transiently transfected MDCK cells. a–c, CLDN16 Y207X does not localize to TJs. Cells were immunostained to detect CLDN16 (HA tag, a; red) and ZO-1 (b, green) and the two images merged (c). Inset, Colocalization of wild-type HA-CLDN16 and ZO-1. d–f, Localization of CLDN16 Y207X to lysosomes. Labeling of CLDN16 Y207X (HA tag, d; red) and the lysosomal marker Lamp-2 (e, green). Yellow in the merged image (f) shows colocalization. g–i, Internalization and lysosomal delivery of antiloop antibodies. Live cells were incubated in the presence of antiloop antibody added in the media for 1 h at 37 C and then washed and processed for immunostaining to detect the antiloop antibody (g, green) and Lamp-2 (h, red). Yellow in the merged image (i) indicates colocalization. Note that cells that do not express CLDN16 (arrows) fail to internalize antiloop antibodies. j–l, Blocking clathrin-mediated endocytosis rescues cell surface expression of CLDN16 Y207X. Clathrin-mediated endocytosis was blocked for 1 h at 37 C in the presence of antiloop antibodies. Cells were then washed and immunostained to detect CLDN16 (HA tag, j; red) or the antiloop antibody (k, green). Yellow in the merged image (l) indicates colocalization.

To explore the molecular defect associated with the mutation, we transiently expressed HA-tagged CLDN16 Y207X in renal epithelial MDCK cells. In contrast to wild-type CLDN16, the mutant protein did not colocalize with the TJ marker ZO-1 but showed a prominent intracellular localization (Fig. 1C, a–c). Colocalization experiments with markers for subcellular compartments showed a predominant localization to lysosomes (Fig. 1C, d–f) and, to a lesser extent, the endoplasmic reticulum (ER) (data not shown).

The lysosomal localization suggested that CLDN16 Y207X may be expressed on the plasma membrane transiently and rapidly endocytose. To test this hypothesis, live MDCK cells were incubated with an antibody to the first extracellular loop of CLDN16 at 37 C. Cell expressing CLDN16 Y207X, but not control cells, efficiently internalized antiloop antibodies (Fig. 1C, g–i), indicating that Y207X was exposed on the cell surface, where it was able to bind and internalize the antibodies. The internalized antibodies colocalized with the lysosomal marker Lamp-2 (Fig. 1C, g–i). Consistent with a transient cell surface exposure, inhibition of clathrin-mediated endocytosis via cytosol acidification (12) or hypertonic media (13) (data not shown) led to a dramatic redistribution of CLDN16 Y207X from a predominantly intracellular vesicular to a plasma membrane staining pattern (Fig. 1C, j–l), consistent with a prolonged half-life of Y207X on the cell surface.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion Note Added in Proof References

 
Mutations in CLDN16 invariably result in renal wasting of Mg²⁺ and Ca²⁺, leading to ESRD. In contrast to Mg²⁺, the renal loss of Ca²⁺ can probably be compensated by increased intestinal absorption and mobilization from bone. Thus, patients harboring CLDN16 mutations have normal serum and plasma levels of Ca²⁺. The novel mutation identified here, Y207X, was associated with neonatal hypocalcemic seizures, a phenomenon so far not described in patients harboring mutations affecting the extracellular or transmembrane domains of CLDN16. A review of the medical records for Ca²⁺ levels shows that hypocalcemia is not uncommon during early infancy in patients with CLDN16 mutations and might even lead, as in the case presented here, to hypocalcemic tetany. Interestingly, the other sibling did not experience symptomatic hypocalcemia during infancy, and the mutations reported in the other children were variable, suggesting that infantile hypocalcemia is not a genotype-specific problem but rather reflects the urinary calcium losses due to the disruption of CLDN16 function. Typically, these losses are compensated by enhanced vitamin D-regulated gastrointestinal absorption. Although release of Ca²⁺ from the stores in bone should be taken into account, PTH values are not always helpful in the setting of FHHNC because reduced kidney function leads by itself to secondary hyperparathyroidism. This also holds true for 1,25(OH)₂D₃ levels because its production can be compromised in patients with chronic renal failure. Kang et al. (14) reported on two siblings with elevated 25-OH and 1,25(OH)₂ D₃ levels despite a compromised renal function, suggesting intestinal compensation. In our case, we speculate that the urinary calcium loss resulting from the defect in CLDN16 may expose borderline vitamin D deficiency by overwhelming the gastrointestinal compensation. We therefore propose to consider mutations in CLDN16 in childhood hypocalcemia. This idea is also supported by the study of Weber et al. (5), who encountered patients with neonatal rickets in their collective of FHHNC patients.

We here present two lines of evidence that the Y207X mutant is properly targeted to the plasma membrane, but rapidly endocytosed and delivered to lysosomes: 1) blocking clathrin-mediated endocytosis increases plasma membrane expression of the mutant, and 2) antibodies against the predicted first extracellular loop of CLDN16 become internalized with the mutant, suggesting previous exposure of this loop at the cell surface.

The Y207X truncation is only the second mutation reported to target the C-terminal cytosolic domain of CLDN16. The other one, T233R, disrupts a C-terminal PDZ binding motif that physically interacts with PDZ domains of ZO-1 (7). Interestingly, both mutants show rapid endocytosis and lysosomal staining in MDCK cells, suggesting that the PDZ binding motif-mediated interaction is necessary to retain CLDN16 at the plasma membrane and hence the TJ. This interpretation is consistent with the observation that the association between CLDN16 and ZO-1 increases the reabsorption of divalent cations in renal epithelial cells (16). A fraction of Y207X is also detected in the ER, suggesting that folding may be compromised, and/or ER exit occurs with slower kinetics.

Although both T233R and Y207X are largely absent from the surface of transfected MDCK cells at steady state, young children affected by the T233R or Y207X mutation displayed hypercalciuria, but only one of the patients with the Y207X mutation presented with neonatal tetany. This could be due to individual differences (i.e. vitamin D levels) but may also reflect differences in the efficiency or rates by which the two mutants either exit the ER or undergo endocytosis, recycling, and lysosomal transport. Such variations could determine the number of CLDN16 molecules transiently expressed on the plasma membrane at any given time and lead to different clinical manifestations. Alternatively, depending on the location and nature of the mutation, the capacity of CLDN16 to transport Ca²⁺ could be differentially affected. Direct Mg²⁺ and Ca²⁺ transport measurements for the two mutants will address this issue.

Proper plasma membrane targeting but rapid endocytosis of Y207X and T233R raise the possibility that localized inhibition of endocytosis, for example using sucrose (13), chlorpromazine (17), or small molecule inhibitors of dynamin-1 GTPase activity (18), could provide novel therapeutic approaches for FHHNC patients carrying these mutations. Our findings therefore highlight the relevance of understanding the precise molecular defects of different CLDN16 mutants to develop specific therapeutic approaches.

	Note Added in Proof

Top Abstract Introduction Patients and Methods Results Discussion Note Added in Proof References

 
We recently identified Cldn16 L203X, a third Cldn16 mutation that affects the C-terminal cytosolic domain (15).

	Acknowledgments

 
We thank the family for their cooperation.

	Footnotes

 
This work was supported by the Agency for Science, Technology and Research, Singapore, and by the 6th Framework Programme of the European Union (EuReGene FP6005085).

The authors declare that no conflicts of interest exist.

First Published Online May 16, 2006

¹ D.M. and P.J.K. contributed equally to the work.

Abbreviations: ER, Endoplasmic reticulum; ESRD, end stage renal disease; FHHNC, familial hypomagnesemia with hypercalciuria and nephrocalcinosis; TJ, tight junction.

Received January 30, 2006.

Accepted May 8, 2006.

	References

Top Abstract Introduction Patients and Methods Results Discussion Note Added in Proof References

 

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