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
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Abstract |
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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 exonintron 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
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Introduction |
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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 -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
-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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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 212) and the corresponding exonintron 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), 12 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 221427 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 1642 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 [-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.
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Results |
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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 2; Table 2
). 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 2
A, Table 2
), 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 3
A 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 3
B, 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 3
B, 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 3
B, lane 4).
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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 2B). 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.
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Discussion |
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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 1). 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 2
; Table 2
). The three mutations were confirmed and demonstrated to co-segregate with the disease by using SSCP and/or restriction enzyme analysis (Figures 1
and 3
). 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 2). 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
-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
-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 proteinprotein 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 1). 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.
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Acknowledgments |
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Notes |
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References |
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