1 Department of Pediatrics, Unit of Nephrology, Hacettepe University Faculty of Medicine, Ankara, Turkey, 2 Department of Pediatrics, Philipps University of Marburg and 3 Department of Clinical Biochemistry, University of Bonn, Bonn, Germany
Correspondence and offprint requests to: Nesrin Besbas, MD, Hacettepe University Faculty of Medicine, Department of Pediatric Nephrology, 06100 Sihhiye, Ankara, Turkey. Email: nbesbas{at}hacettepe.edu.tr
Keywords: chloride channel 5 (ClC-5); CLCN5 gene; Dent's disease; metabolic alkalosis
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Introduction |
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Inherited disorders that manifest hypokalaemic metabolic alkalosis, such as the BartterGitelman syndrome or the hyperprostaglandin E syndrome (also referred to as antenatal Bartter's syndrome), are caused by the malfunction of renal tubular electrolyte transporters or ion channels. The hyperprostaglandin E syndrome is linked to the dysfunction of the sodiumpotassiumchloride co-transporter (NKCC2) [7] or the renal outer medullary potassium channel (ROMK) [8]. The cardinal features of the syndrome are its antenatal onsetwith polyhydramnios due to fetal polyuria, isothenuria and medullary nephrocalcinosis. When associated with sensorineural deafness or autosomal dominant hypocalcaemia, the hyperprostaglandin E syndrome is due to mutations in barttin, a ß subunit of voltage-gated chloride channels [9], and the calcium-sensing receptor CaSR [10]. BartterGitelman syndrome is linked to mutations in the basolateral chloride channel (ClC-Kb) [11] or in the sodiumchloride co-transporter (NCCT) [12]. The course of this disease is usually milder, mimicking chronic use of thiazides. ROMK, NKCC2 and NCCT mutations usually have uniform clinical presentations, whereas mutations in CLCNKB, encoding ClC-Kb, occasionally lead to phenotypic overlaps with the ROMK/NKCC2 cohort.
This study describes the first case of Dent's disease due to a loss-of-function mutation in the CLCN5 gene, R347X, associated with a Bartter-like syndrome that is characterized by hypokalaemic metabolic alkalosis and secondary hyper-reninaemic hyperaldosteronism.
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Case |
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Gene analysis in the patient included the genes coding for: (i) the chloride channel 5 (CLCN5); (ii) the thiazide-sensitive Na+/Cl co-transporter (SLC12A3); (iii) the renal chloride channel (CLCNKB); (iv) the NaK2Cl co-transporter (SLC12A1); and (v) the renal outer medullary K+ channel (KCNJ1). In order to detect the mutation responsible for our patient's disease, we initially amplified all exons (with their corresponding splice sites) of the genes encoding ClC-5, NCCT and ClC-Kb. Sequence analysis of the resultant polymerase chain reaction (PCR) products revealed only one deviation from the wild type in the PCR product corresponding to the CLCN5 exon 8. The sequence variation detected in this sample turned out to be a hemizygous C1330T transition which codon 347 (CGA: arginine) changed to a premature stop codon. [Nucleotide numbering is according to the cDNA (accession no. NM_000084) with the A of the start-ATG at nucleotide 292.] This C1330T substitution was not present in samples from 50 Caucasian controls (25 males and 25 females = 75 alleles), and family analysis revealed that the patient had inherited this mutant allele from his asymptomatic carrier mother.
To elucidate further the aetiology of the patient's phenotype, sequence analysis was also performed for his genes encoding ROMK and NKCC2; however, no abnormalities could be detected. Aside from known CLCNKB polymorphisms (see single nucleotide polymorphism data listed in the Ensemble Human Genome Browser), we also detected two novel intronic variants in the patient: in intron 5, a homozygous C to T transition was observed at position 5 of the acceptor splice site of exon 6 (IVS5 5), and the intron 10 position +13 (IVS10 +13) was found to be heterozygous (G/C). Since aberrant CLCNKB mRNA splicing would provide an explanation for the phenotype observed in our patient, these variants were tested not only for their occurrence in a panel of normal control subjects, but also for their potential to affect normal splicing. The initial restriction analysis of the respective PCR products showed both variants to be true polymorphisms: we observed allelic frequencies of 0.78 (T) and 0.22 (C) in IVS5 5, and of 0.63 (C) and 0.37 (G) for the IVS10 +13 variant. We then analysed CLCNKB mRNA transcripts from subjects homozygous for each of these polymorphisms. However, in all cases, the amplification of products attributable to exons 57 and 912/13 revealed only the normal message and provided no evidence of exon skipping or cryptic splice site usage (data not shown).
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Discussion |
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Inherited disorders that manifest hypokalaemic metabolic alkalosis such as the BartterGitelman and hyperprostaglandin E syndromes are caused by the malfunction of renal tubular electrolyte transporters or ion channels. All variants share several clinical characteristics, including renal salt wasting, hypokalaemic metabolic alkalosis and hyper-reninaemic hyperaldosteronism. Our patient presented with hypokalaemic metabolic alkalosis associated with hyper-reninaemic hyperaldosteronism, hypercalciuria and nephrocalcinosis, but without antenatal history, thereby suggesting Bartter's syndrome. However, we also identified LMWP, which is not a feature of Bartter's syndrome, but which is the most consistent laboratory finding in Dent's disease. This raises the question of whether or not both Bartter's syndrome and Dent's disease might be present in the same patient.
It is known that mutations in NCCT, ROMK and NKCC2 usually have uniform clinical presentation, whereas mutations in CLCNKB, encoding ClC-Kb, occasionally lead to phenotypic overlaps with the ROMK/NKCC2 cohort. For this reason, we searched not only for CLCN5 mutations but also for defects in the genes encoding NCCT, ClC-Kb, ROMK and NKCC2. However, apart from the identification of the CLCN5 exon 8 mutation (R347X), we did not detect any mutations in these genes from our patient. We only observed two novel intronic CLCNKB nucleotide substitutionswhich were found to represent neutral polymorphisms that do not interfere with normal proper splice site recognition. Since the IVS10 +13G>C variant does not affect the donor motif of exon 10, and no consensus value (CV) could be predicted, these findings are consistent with the CV obtained for the acceptor splice site variation in intron 5 (IVS5 5C>T) in our patient. Here, nearly identical values could be calculated (type C, ctcggatcccccag A, CV = 0.813; type T, ctcggatcctccag A, CV = 0.823; where the substitution is underlined, and indicates the splice site, with the exonic A in upper case); and the T value observed in our patient was even better.
Other rare variants of inherited hypokalaemic salt-losing tubulopathies could be excluded in our patient clinically. The onset of the disorder was after infancy, which is unusual for the hyperprostaglandin E syndrome. In addition, he did not have a sensorineural deafness, which is invariably linked to barttin mutations, and a CaSR defect would be unlikely due to the absence of hypocalcaemia. However, compared with the X-linked CLCN5 gene, the mutation might have been missed due to the failure to amplify the respective PCR product from one of the two alleles in the tested autosomal genes (SLC12A1, SLC12A3, KCNJ1 and CLCNKB). Also, the failure to detect mutation could be due to the fact that the regulating elements of these genes (elements located in intronic regions or proximal or distal to these genes) either were not investigated or the amplification technique will not identify deletions or inversions, or both. On the other hand, the absence of mutations in the patient we studied does not exclude the possibility that any of these genes could be implicated in other patients with phenotypes resembling the BartterGitelman or hyperprostaglandin E syndromes.
The R347X mutation has been reported previously in two Japanese patients, where it was associated with a milder phenotype than we observed in our patient [3,4]. These Japanese children had LMWP and slightly increased calcium excretion, but no nephrocalcinosis. Younger age, hypokalaemic hypochloraemic metabolic alkalosis, hypophosphataemia, nephrocalcinosis and nephrolithiasis without osteopenia and rickets are the main features which are different in our case compared with these two patients (Table 1). A third report described an American patient with the R347X mutation [5]; and here, although no clinical details were given, a fuller phenotype of Dent's disease was manifest. This reflects the general observation that a single CLCN5 mutation can be associated with a range of phenotypes.
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Chloride efflux across the basolateral membrane of the thick ascending limb can occur mainly via the ClC-Kb channel, but also via other chloride channels, such as the highly homologous ClC-Ka channel, CFTR, and ClC-5. Also, members of the potassium chloride co-transporter family are localized in this nephron segment. Hence, a defect in the activity of ClC-Kb is expected to reduce NaCl reabsorption in these tubule segments, but does not abolish salt reabsorption completely. Defects in any of these proteins would impair net NaCl reabsorption in the thick ascending limb, and thereby increase NaCl delivery to more distal nephron segmentswhose consequence is salt-wasting volume contraction and stimulation of the reninangiotensinaldosterone axis, which leads to hypokalaemic metabolic alkalosis. The metabolic alkalosis of our patient was most probably due to this mechanism.
It has long been known that Bartter's syndrome may be secondary to other familial disorders that affect the kidneys, such as nephropathic cystinosis, KearnsSayre syndrome, Menke's kinky hair syndrome and familial renal dysplasia. Drugsthe chronic use of diuretics and aminoglycosides and chemotherapycan mimic BartterGitelman syndrome. Since none of them was in use in our patient at the time of diagnosis, we excluded these secondary causes of that syndrome.
In conclusion, in metabolic alkalosis accompanied by Dent's disease, one or more additional mutations in one or more other genes may provoke a disorder that phenocopies other tubular disorders. The co-existence of hypokalaemic metabolic alkalosis and LMWP associated with renal stone disease should prompt clinicians to search for Dent's disease.
Conflict of interest statement. None declared.
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References |
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