Novel truncating mutations in the ClC-5 chloride channel gene in patients with Dent's disease

Irma Carballo-Trujillo1, Victor Garcia-Nieto1,2, Francisco J. Moya-Angeler3, Montserrat Antón-Gamero4, Cesar Loris5, Sebastián Méndez-Alvarez1 and Felix Claverie-Martin1,

1 Research Unit and 2 Pediatric Nephrology Unit, Nuestra Señora de Candelaria University Hospital, Santa Cruz de Tenerife, 3 Department of Pediatrics, Virgen Macarena University Hospital, Sevilla, 4 Department of Pediatrics, Reina Sofia University Hospital, Cordoba and 5 Department of Pediatrics, Miguel Servet Hospital, Zaragoza, Spain



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Dent's disease is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and eventual renal failure. The disease is caused by mutations in the X-linked chloride channel CLCN5 gene, which encodes a 746-amino-acid protein expressed in renal tubules. These mutations have been reported in unrelated families from the UK, USA, Japan and other countries. We were interested in identifying additional mutations in the CLCN5 coding region of Spanish patients with Dent's disease.

Methods. Five patients from three unrelated Spanish families were studied. Leukocyte genomic DNA from patients and their relatives was used with CLCN5-specific primers for polymerase chain reaction amplification of the coding region and exon–intron boundaries. Amplified products were analysed by single-strand conformational polymorphism analysis, DNA sequencing and restriction enzyme analysis.

Results. Low-molecular-weight proteinuria and hypercalciuria were detected in all the patients, nephrocalcinosis in two patients, and rickets or osteopenia in three patients. We identified three new CLCN5 mutations consisting of two nonsense mutations, Leu433Stop and Arg718Stop, and an insertional frameshift mutation, 65insT, which results in a stop at codon 98. These three mutations predict truncated ClC-5 proteins that, respectively, lack 314, 649 and 28 amino acids at the carboxy terminus, and are likely to result in loss of function. These mutations were shown to co-segregate with the disease in each of the three families.

Conclusions. Our study is the first to characterize mutations in the CLCN5 gene in Spanish patients with Dent's disease and expands the spectrum of CLCN5 mutations associated with this disease.

Keywords: chloride channel 5 (ClC-5); CLCN5 gene; Dent's disease; hypercalciuria; mutation; X-linked renal disease



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Dent's disease is now the generally accepted name for a group of hereditary tubular disorders including X-linked recessive nephrolithiasis with renal failure, X-linked recessive hypophosphataemic rickets and idiopathic low-molecular-weight proteinuria [1,2]. Mutations in the renal chloride channel gene, CLCN5, have been shown to be the cause of this disease [1,3]. The CLCN5 gene is located on the short arm of the X chromosome (Xp11.22) [4]. Dent's disease is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, eventual renal failure and in some cases rickets or osteomalacia [2]. The disease tends to present in childhood or early adult life, and males are more severely affected than females. The selective loss of low-molecular-weight proteins points to a defect of the renal proximal tubule of the kidney, where filtered proteins are normally reabsorbed by endocytosis.

The CLCN5 gene has been cloned and characterized; it contains 11 coding exons with an open reading frame of 2238 nucleotides encoding a protein of 746 amino acids [5]. This protein, referred to as ClC-5, is highly expressed in the kidney [5], and belongs to the family of voltage-gated chloride channel (ClC) proteins [6]. The three-dimensional crystal structure of two bacterial ClC chloride channels has been described recently [7]. This structure reveals two identical pores, each formed by a separate subunit contained within a homodimeric membrane protein. Each monomer contains 18 {alpha}-helices and has an internal repeat structure in an antiparallel orientation. A comparison of the new helix nomenclature with the previously used D1 to D12 nomenclature is given in Jentsch et al. [6]. There are nine different ClC proteins in mammals, some of which are plasma membrane channels and the others are thought to reside predominantly in intracellular membranes. Several ClC channels are associated with different human diseases; mutations in CLCN1 lead to myotonia congenita, mutations in CLCNKB lead to a form of Bartter's syndrome, mutations in CLCN7 lead to severe juvenile osteopetrosis, and mutations in CLCN5 lead to Dent's disease [6]. Recent studies have shown that ClC-5 is intracellularly located in subapical endosomes of kidney epithelial cells lining the proximal tubules, the thick ascending loop of Henle, and {alpha}-intercalated cells of the collecting ducts [8,9]. These studies have also demonstrated that ClC-5 is co-localized with vacuolar H+-ATPases and with endocytosed ß2-microglobulin in intact proximal tubule cells. These findings suggest that the intracellular ClC-5 chloride channel provides an electrical shunt for the electrogenic proton pump, which is required for the acidification of the organelles involved in the endocytosis of low-molecular-weight proteins [8]. Furthermore, ClC-5 knockout mice exhibit a phenotype similar to that of Dent's disease with low-molecular-weight proteinuria associated with impaired proximal tubule reabsorption of proteins [10,11]. This evidence provides confirmation of the crucial role of ClC-5 in proximal tubule endocytosis. However, the mechanisms by which CLCN5 mutations result in hypercalciuria and other tubular abnormalities remain to be elucidated. The identification of additional mutations may help in these studies.

In this study, we analysed the coding region of the CLCN5 gene in five Spanish patients with Dent's disease and identified three new mutations consisting of two nonsense mutations, and an insertional frameshift mutation. These three mutations predict truncated ClC-5 proteins and are likely to result in loss of function.



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 Subjects and methods
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Patients
Five patients with Dent's disease, from three unrelated Spanish families (F1, F2, F3), were investigated. The clinical and biochemical data of the five affected members are summarized in Table 1Go. The five patients, all males, had low-molecular-weight proteinuria and hypercalciuria, two had nephrocalcinosis, three had rickets or osteopenia, four had reduced tubular reabsorption of phosphate, and four had urinary concentrating ability defect. The available data on acid–base status indicated that they were basically normal. During the follow-up period, four patients showed a slight decreased of glomerular filtration rate (GFR). A history of Dent's disease could be established in one of the families, while in the other two, no families members were available for study to establish an inherited basis for the disease. Clinical information of the carrier mothers was not available. This study was approved by the Ethics Committee of Nuestra Señora de Candelaria University Hospital (Santa Cruz de Tenerife, Spain). All patients' parents and adult patients gave informed consent to participate in the study.


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Table 1.  Phenotypical evaluation of five Spanish patients with Dent's disease

 
Patient F1-II.1 was a 20-year-old male who at the age of 8 years had mild proteinuria (520 mg/dl), initially found at a routine laboratory test. Further analysis had shown low-molecular-weight proteinuria (32 500 µg ß2-microglobulin/l), hypercalciuria (9.4 mg/kg/day), a reduced tubular reabsorption of phosphate (TRP, 75%), and an impaired urinary concentrating ability (maximum urinary osmolality, 540 mOsm/kg). At 13 and 16 years of age, evaluation of bone mineral density (BMD) had shown demineralization (Z-scores -2.5 and -3.8, respectively). At age 19 years, a progressive decrease in creatinine clearance (61 ml/min/1.73 m2) had been observed. A test had revealed normal renal function in this patient's parents.

Patient F2-II.1 was a 30-month-old boy with proteinuria (102 mg/dl) that was detected in a routine urine test. Laboratory examinations showed high levels of urinary ß2-microglobulin (154 660 µg/l), hypercalciuria (9.6 mg/kg/day), a reduced TRP (65.8%) and normal GFR. There was no family history of renal disease. His brother, patient F2-II.2, then at age 10 years, was also examined and showed proteinuria (84 mg/dl), consisting of low-molecular-weight proteinuria (66 230 µg ß2-microglobulin/l), hypercalciuria (9.7 mg/kg/day), decrease in creatinine clearance (77 ml/min/1.73 m2), and reduced TRP (68.6%). There was no evidence of nephrocalcinosis, nephrolithiasis or osteopenia in these two brothers.

Patient F3-III.1 was an asymptomatic 10-year-old boy belonging to family 3. His two maternal uncles (F3-II.1 and F3-II.2) have Dent's disease. Proteinuria was detected at a routine urine test. Subsequent laboratory examinations showed proteinuria (260 mg/dl) with high levels of urinary ß2-microglobulin (80 000 µg/l), hypercalciuria (13.3 mg/kg/day), decrease in creatinine clearance (77 ml/min/1.73 m2) and diminished urinary concentrating ability (maximum urinary osmolality, 576 mOsm/kg). The evaluation of BMD showed a slight osteopenia. Both renal ultrasound and percutaneous kidney biopsy showed nephrocalcinosis.

Patient F3-II.1 was the maternal uncle of patient F3-III.1. At age 8 months, he had had a urinary infection and the examination had shown proteinuria and rickets. At age 5 years, he had tubular proteinuria, hypercalciuria (39 mg/kg/day), and the urine concentration ability (740 mOsm/kg) and TRP (68%) were reduced. At age 22 years, he had low-molecular-weight proteinuria (ß2-microglobulin 51 200 µg/l), renal osteodystrophy and radiological and sonographic nephrocalcinosis. Currently (age 25 years), this patient has moderate chronic renal failure. His clinical record shows that his brother (F3-II.2), at age 26 years, had had renal failure and started haemodialysis treatment, and at age 31 years had received a kidney transplant.

DNA amplification by polymerase chain reaction
Genomic DNA of all affected individuals, available family members and unrelated normal individuals was extracted from whole blood using the Qiagen DNA Blood Mini kit (Qiagen GmbH, Hilden, Germany). Thirteen pairs of primers [3] were used to amplify the coding sequences (exons 2–12) and the corresponding exon–intron boundaries of CLCN5. Primers were synthesized by TIB Molbiol (Berlin, Germany). Polymerase chain reaction (PCR) was performed by adding 1 µl of DNA sample to a mixture containing 1x (NH4)2SO4 reaction buffer (Bioline, UK), 1–2 mM MgCl2, 0.2 mM of each dNTP, 25 pmol of each pair of primers, and 1.5 U of Taq DNA polymerase (Bioline, UK), in a final volume of 50 µl. Reactions were carried out in a GeneAmp PCR system 9700 (PE Applied Biosystems, CA) with the following thermal cycling profile: an initial denaturation step at 94°C for 5 min, followed by 35 cycles of amplification (45 s at 94°C; 45 s at 56, 58 or 59°C, depending on the pair of primers; and 1 min at 72°C), and an extension at 72°C for 7 min. PCR products were examined by electrophoresis on 1.5% (w/v) agarose gels and ethidium bromide staining. The size of PCR products were 221–427 bp, as expected.

Single-strand conformation polymorphism analysis
The CLCN5 gene was screened for mobility shifts by single-strand conformation polymorphism (SSCP) analysis using the DCode electrophoresis system (Bio-Rad Laboratories, Hercules, CA). The PCR products were denatured at 95° C for five minutes and electrophoresed under non-denaturing conditions on 10, 12.5, 14 and 16% (w/v) polyacrylamide gels at 10, 15 and 20°C at 200 V for 16–42 h. The band patterns were visualized by the silver staining method. Amplified products from genomic DNA of unrelated normal individuals were used as controls in the SSCP analysis. Mobility shift of single-strand DNA from the normal pattern indicated the presence of a possible mutation.

DNA sequence analysis
The DNA sequence of PCR products with SSCP bands that differed from the normal controls was determined by the dideoxynucleotide chain termination method. The PCR-amplified DNA was isolated by electrophoresis on a 1.5% agarose gel and ligated into vector pGEM-T-easy as described in the technical manual (Promega Corporation, Madison, WI). Positive clones were selected and plasmid DNA was purified with the Qiagen Plasmid kit (Qiagen). Both strands of the DNA fragments were sequenced using the Sequenase version 2.0 DNA sequencing kit (Amersham Life Science Inc., IL), SP6 and T7 primers, and [{alpha}-35S]dATP (Nucliber SA, Madrid, Spain). Reaction products were resolved on 6% polyacrylamide gels containing 7 M urea. Gels were dried and DNA bands were visualized by autoradiography. DNA sequence abnormalities were confirmed by analysis of PCR products from three independent amplifications and by either restriction endonuclease analysis of PCR products or by SSCP (see below). We also sequenced PCR fragments from normal individuals as controls.

Restriction fragment length polymorphism analysis
To facilitate the detection of the mutations found in families 1 and 3, PCR-amplified DNAs of exons 8 and 12 from patients, family members and normal individuals were digested with restriction enzymes Tru9I (restriction site, T/TAA; Roche Diagnostics GmbH, Mannheim, Germany) and HphI (restriction site, GGTGAN8/7/N; MBI Fermentas, Vilnius, Lithuania), respectively. Restriction digestions with Tru9I and HphI were carried out at 65 and 37°C, respectively, under the conditions indicated by the suppliers. The oligonucleotide primers used for amplification were 8.2F and 8.2R, and 12F and 12R [3], respectively. The restriction products were electrophoresed together with DNA molecular weight marker XIV (100 bp ladder, Roche Diagnostics) on 1.5% (w/v) agarose (HphI digestion) or 4% (w/v) NuSieve GTG agarose (Tru9I digestion) (FMC BioProducts, Rockland, ME) gels, and the DNA was stained with ethidium bromide.



   Results
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 Subjects and methods
 Results
 Discussion
 References
 
Detection of mutations by SSCP analysis
In order to detect and characterize the mutations responsible for the disease in our five patients (Table 1Go), genomic DNA from each patient was amplified by PCR with 13 pairs of primers encompassing all coding exons of the CLCN5 gene. The PCR products were screened for mutations by SSCP analysis as described in Subjects and methods. Bands with abnormal mobility were identified in the PCR products corresponding to exons 8, 3 and 12 of patients from families 1, 2 and 3, respectively (Figure 1Go). The difference in SSCP patterns in exons 8, 3 and 12 were best observed using 14, 16 and 10% (w/v) polyacrylamide, respectively, and a running temperature of 15°C. DNA from the patients (F1-II.1, F2-II.1, F2-II.2, F3-II.1 and F3-III.1) and from F3-II.2 revealed the mutant alleles only, whereas DNA from unaffected fathers (F1-I.1, F2-I.1 and F3-II.4), unaffected daughter (F2-II.3), unaffected grandfather (F3-I.1), and normal unrelated individuals (N) revealed the wild-type alleles only. The PCR products of the patients' mothers (F1-I.2, F2-I.2, F3-I.2 and F3.II.3) showed both the normal and the abnormal bands (Figure 1Go). No other abnormal bands were detected with the 10 remaining exons of each patient (results not shown).



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Fig. 1.  Detection of CLCN5 mutations in exons 8, 3 and 12 by SSCP analysis. The PCR amplification of exon 8 was carried out with primers 8.2F and 8.2R [4]. PCR products were run on 14, 16 and 10% (w/v) polyacrylamide gels, respectively, at 15°C and at 200 V for 36, 42 and 16 h, respectively. The DNA was detected by silver staining. Abnormal SSCP bands (arrows), corresponding to mutant alleles, could be distinguished from normal bands (arrow heads), corresponding to the wild-type alleles. (A) Exon 8 from family 1: affected son (II.1), mother (I.2) and unaffected father (I.1). (B) Exon 3 from family 2: affected sons (II.1 and II.2), unaffected daughter (II.3), unaffected mother (I.2), unaffected father (I.1), and unrelated normal individual (N). (C) Exon 12 from family 3: affected son (III.1), mother (II.3), affected maternal uncles (II.1 and II.2), grandmother (I.2), unaffected grandfather (I.1) and unaffected father (II.4). The corresponding pedigree is shown below each gel. Symbols: (•) female carrier, ({blacksquare}) affected male, ({circ}) unaffected female and ({square}) unaffected male.

 

Characterization of mutations
PCR fragments showing abnormal mobility were cloned and sequenced. The DNA sequences revealed the presence of three new mutations in the CLCN5 gene that consisted of two nonsense mutations and one insertional mutation (Figure 2Go; Table 2Go). The point mutation found in exon 8 of patient F1-II.1 consisted of a base change, in position 1589 of the CLCN5 cDNA (numbering as in [5]), of a T to a G that changed the codon Leu433 (TTA) to a stop codon (TGA) (Figure 2GoA, Table 2Go), predicting synthesis of a truncated ClC-5 protein shortened by 314 amino acids. This mutation, Leu433Stop, abolishes a Tru9I restriction site, and digestion with this enzyme was used as a rapid test to confirm and detect the mutation in other members of this family (Figure 3GoA and B). Genomic DNA was amplified with primers 8.2F and 8.2R [4], giving a PCR product of 321 bp. After digestion with Tru9I, normal individuals had three fragments of 76, 131 and 114 bp (Figure 3GoB, lanes 2 and 3), while patient F1-II.1 was missing the two bands of 131 and 114 bp, and had an altered fragment of 245 bp. (Figure 3GoB, lane 5). The patient's mother was heterozygous for this mutation; digestion of her DNA with Tru9I gave four fragments of 76, 131, 114 and 245 bp (Figure 3GoB, lane 4).



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Fig. 2.  Characterization of mutations in exons 8, 3 and 12 of CLCN5 by DNA sequence analysis. Partial nucleotide sequences of PCR amplified DNAs from exons 8 (A), 3 (B) and 12 (C) are shown. Both the mutant and the wild-type sequences are shown. The mutations are indicated by arrows on the autoradiography, and by asterisks on the nucleotide sequence. The predicted amino acid sequences are also shown. (A) A single T to G base change converting codon Leu433 to a termination codon TGA was observed in the sequence of exon 8 from patient F1-II.1. (B) An insertion of a T in codon 65 producing a frameshift is found in exon 3 of patient F2-II.1. (C) A single C to T base change converting codon Arg718 to a stop codon was detected in exon 12 of patient F3-III.1.

 

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Table 2.  CLCN5 mutations found in Spanish patients with Dent's disease

 


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Fig. 3.  Detection of CLCN5 mutations in families 1 and 3 by restriction enzyme analysis. (A) The point mutation (T to G) found in exon 8 from patient F1-II.1 resulted in the loss of a Tru9I restriction enzyme site (T/TAA), indicated by vertical arrows. PCR amplification with primers 8.2F and 8.2R and digestion with Tru9I would result in three DNA fragments of 76, 131 and 114 bp from the normal sequence, but only two fragments of 76 and 245 bp from the mutant sequence. (B) Restriction digestion products from family 1 were separated by electrophoresis on a 4% (w/v) NuSieve agarose gel. Lane M, molecular weight marker (100 bp ladder); lane 1, undigested PCR fragment; lanes 2 and 3, normal male controls (N1 and N2); lane 4 patient's mother (F1-I.2); lane 5, patient F1.II.1. (C) The PCR fragment corresponding to exon 12 contains only one HphI restriction enzyme site (GGTGAN8/7/) (indicated by a vertical arrow). The C to T transition found in patient F3-III.1 resulted in the gain of a HphI restriction site. PCR amplification and HphI digestion would result in one fragment of 233 bp and one fragment of 18 bp from the normal sequence, but three fragments of 87, 18 and 146 bp from the mutant sequence. (D) Restriction digestion products from family 3 were separated by electrophoresis on a 1.5% (w/v) agarose gel. Lane M, molecular-weight marker (100 bp ladder); lane 1, undigested PCR fragment; lanes 2 and 3, normal male controls (N1 and N2); lane 4, patient's father (F3-II.4); lane 5, patient's grandfather (F3-I.1); lane 6, patient's mother (F3-II.3); lane 7, patient's grandmother (F3-I.2); lanes 8, 9 and 10, patients F3-III.1, F3-II.1 and patient's maternal uncle (F3-II.2), respectively. The 18 bp fragment is not shown. Wt, wild-type; m, mutant; bp, base pairs.

 
DNA sequencing of abnormal exon 12 from patient F3-III.1 revealed a C to T transition in position 2443, converting codon Arg718 (CGA) to a stop codon (TGA) (Figure 2GoC; Table 2Go). This mutation would result in synthesis of a truncated ClC-5 protein that lacks 28 amino acids at its carboxy terminus. This alteration also generates an HphI restriction site in exon 12, and therefore we used the digestion with HphI to confirm the base change on PCR product amplified from the patient and his relatives (Figure 3GoC and D). Genomic DNA was amplified with primers 12F and 12R [3] resulting in a product of 251 bp. There was one HphI restriction site in the PCR products from normal individuals (Figure 3GoD, lanes 2 and 3) and from the patient's father (F3-II.4) and grandfather (F3-I.1) (Figure 3GoD, lanes 4 and 5), resulting in a fragment of 233 bp and a small fragment of 18 bp (data not shown). When the PCR products from patient F3-III.1 and his two maternal uncles (F3-II.1 and F3II.2) were analysed, the 233 bp band was absent and two new bands of 87 and 146 bp appeared (Figure 3GoD, lanes 8–10). The patient's mother (F3-II.3) and maternal grandmother (F3-I.2) were heterozygous with both wild-type and mutant alleles; digestion of their PCR products with HphI resulted in four fragments of 233, 87 and 146 bp (Figure 3GoD, lanes 6 and 7) and 18 bp (data not shown).

The sequence of the exon 3 band with abnormal mobility (from patients F2-II.1 and F2-II.2) showed a single T insertion in codon 65 (Figure 2GoB). This mutation, 65insT, leads to a premature stop codon due to a reading frameshift. The resulting truncated protein, of 97 amino acids, would have 32 amino-acid changes at the carboxy terminus and would lack 649 amino acids of the normal ClC-5 protein.

We used the Tru9I and HphI restriction analysis (for mutations Leu433Stop and Arg718Stop, respectively) and SSCP analysis (for 65insT, as this mutation does not create or abolish any restriction site) to ensure that the abnormal DNA sequences were not common polymorphisms. The absence of each of the three sequence abnormalities in 120 alleles from 80 unrelated healthy Spanish individuals (40 women and 40 men; data not shown) established that they were mutations and not polymorphisms that would be expected to occur in >1% of the population.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We studied five male patients with the typical symptoms of Dent's disease from three unrelated Spanish families (Table 1Go). The five patients had low-molecular-weight proteinuria and hypercalciuria, two had nephrocalcinosis, three had rickets or osteopenia, three had slightly decreased glomerular filtration rate, and the brother of one of the patients had terminal renal failure. All our patients showed microhaematuria, which, in general, is uncommon in Dent's disease patients, but has been reported in some cases [12]. A history of Dent's disease could be established in one of the families, while in the other two families members were not available for the study to establish an inherited basis for the disease.

SSCP analysis has proved to be very effective in detecting point mutations in the CLCN5 gene [3,13,14]. We used this technique to screen for mutations in the 11 coding exons of CLCN5 in our patients and their relatives (Figure 1Go). DNA bands with abnormal mobility were sequenced. Three new mutations were identified which consisted of two nonsense mutations creating premature stop codons (Leu433Stop and Arg718Stop) and a single nucleotide insertion (65insT) that produces a frameshift and also results in premature termination of translation (Figure 2Go; Table 2Go). The three mutations were confirmed and demonstrated to co-segregate with the disease by using SSCP and/or restriction enzyme analysis (Figures 1Go and 3Go). Furthermore, the absence of these abnormalities in 120 alleles from 80 unrelated normal individuals (40 females and 40 males) demonstrated that they were not common polymorphisms (results not shown). Mutation Arg718Stop and previously described mutations Arg28Stop, Arg34Stop, Arg347Stop, Arg637Stop, Arg648Stop, Ser244Lue, and Arg704 [1,12,15,16] involve a C to T transition that occurs at a CpG site, which is the most common site of methylation and represents a potential hot spot for mutations.

The two nonsense mutations, Leu433Stop and Arg718Stop, and the insertional frameshift mutation, 65insT, predict truncated ClC-5 channels that lack, respectively, 314 amino acids (42%), 28 amino acids (4%), and 649 amino acids (87%) from the carboxy terminus (Table 2Go). In the case of the insertional mutation, the ClC-5 sequence will stop at amino acid 64 and will be followed by a missense peptide of 32 amino acids (Trp-Ala-Phe-Ile-Arg-Phe-Val-Ser-Trp-Phe-Asp-Arg-His-Leu-Cys-Ser-Leu-Asp-Asp-Arg-Leu-Lys-Arg-Arg-Tyr-Met-His-Arg-Gly-Ile-Leu-Val). The truncated protein predicted for mutation Leu433Stop would lack the hydrophobic region at the carboxy terminus, composed of five {alpha}-helices (M, N, O, P and Q) that cross the membrane several times (domains D9 to D12 in the previous nomenclature [6]) and the intracellular {alpha}-helix R, and the two conserved CBS domains. While the truncated ClC-5 protein predicted for mutation Arg718Stop would lack part of the carboxy terminus including one of the two conserved CBS domains (CBS2). The functional role of CBS domains, which are found in CLC chloride channels and other proteins, is not known, but they could be involved in protein–protein interactions [17]. The fact that a short deletion like Arg718Stop resulted in all the severe symptoms of the disease suggests essential domains in the very carboxy terminus of ClC-5. An assessment of some of the ClC-5 truncating mutations (Arg704Stop, Arg648Stop, Trp279Stop and Arg347Stop) in the Xenopus oocyte expression system has revealed that they produce an abolition of chloride currents [1,15]. Therefore, the three new mutations described here are likely to result in a loss of ClC-5 channel function.

Different CLCN5 mutations associated with Dent's disease have been reported in families from the USA, Canada, UK, Italy, France, India and Japan [1,3,1215,1820]. The cases reported in Southern Europe are relatively few. The identified mutations include nonsense mutations, missense mutations, splice site mutations and deletional and insertional mutations. Approximately 70% of the CLCN5 mutations are likely to result in truncated or absent ClC-5 channels. Most of the mutations are scattered over the CLCN5 coding region, and a correlation between genotype and phenotype has not been established. It is interesting to note that of the three mutations we identified, the mutation in patients F2-II.1 and F2-II.2, 65insT, generated the most truncated ClC-5 protein (87% protein loss), although the phenotype of these patients was not as severe (i.e. absence of nephrocalcinosis, rickets or osteopenia) as that of the other three patients (42 and 4% protein loss, respectively) (Table 1Go). However, a correlation between the length of the truncation and the severity of the disease is hard to establish since truncated proteins are generally unstable. The differences in phenotypes could also be ascribed to unidentified environmental factors or other genetic factors [2]. Functional studies of these mutations in a heterologous system will prove the severity of the mutations and the function of the lacking regions of the protein.

In conclusion, we have identified three new CLCN5 mutations that predict structurally significant alterations of the ClC-5 channel and are therefore likely to result in a loss of function. Our study is the first to characterize mutations in the CLCN5 gene in Spanish patients with Dent's disease, and the results expand the spectrum of CLCN5 mutations associated with this renal tubulopathy.



   Acknowledgments
 
We thank Amable Aguirre Figueras and Alicia Gómez Coello for help with the DNA sequencing, and the patients and their families for their participation. This work was supported by grants 98/1179 and PI1997/051 from the Fondo de Investigación Sanitaria (Spain) and from the Consejería de Educación, Cultura y Deportes (Canarian Autonomous Government), respectively, to F.C.M. and V.G.N., and by Sociedad Canaria de Pediatría. It was presented in part at the 9th European Symposium on Urolithiasis (13–15 September 2001, Rotterdam, The Netherlands). S.M.A. was supported by the Fondo de Investigación Sanitaria (Spain), contract 99/3060.



   Notes
 
Correspondence and offprint requests to: Dr Félix Claverie-Martín, Unidad de Investigación, Laboratorio de Biología Molecular, Hospital Nuestra Señora de Candelaria, Carretera del Rosario, s/n, 38010 Santa Cruz de Tenerife, Spain. Email: fclamar{at}gobiernodecanarias.org Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 23. 7.02
Accepted in revised form: 12.11.02