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Novel Mutations of the Chloride Channel Kb Gene in Two Japanese Patients Clinically Diagnosed as Bartter Syndrome with Hypocalciuria

Department of Pediatrics (S.F., M.Hig., T.O.), Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0125, Japan; Department of Pediatrics (M.Hir.), Nishibeppu National Hospital, Ohita 874-0833, Japan; and Department of Pediatrics (M.A.), Ohtemae Hospital, Osaka 540-0008, Japan

Address all correspondence and requests for reprints to: Dr. Takao Ohta, Department of Pediatrics, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa, 903-0125 Japan. E-mail: tohta{at}med.u-ryukyu.ac.jp.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Hypokalemic metabolic tubulopathy, such as in Bartter syndrome and Gitelman syndrome, is caused by the dysfunction of renal electrolyte transporters. Despite advances in molecular genetics with regard to hypokalemic metabolic tubulopathy, recent reports have suggested that the phenotype-genotype correlation is still confusing, especially in classic Bartter and Gitelman syndromes. We report here two Japanese patients who suffered from clinically diagnosed classic Bartter syndrome but who presented hypocalciuria. Hypocalciuria is generally believed to be a pathognomonic finding of NCCT malfunction. To better understand the genotype-phenotype correlation in these two cases, we screened four renal electrolyte transporter genes [Na-K-2Cl cotransporter (NKCC2), renal outer medullary K channel (ROMK), Cl channel Kb (ClC-Kb), and Na-Cl cotransporter (NCCT)] by the PCR direct sequencing method. We identified three ClC-Kb allelic variants, including two new mutations (L27R and W610X in patient 1 and a G to C substitution of a 3' splice site of intron 2 and W610X in patient 2). We did not find any mutations in the other three genes. Our present data suggest that some ClC-Kb mutations may affect calcium handling in renal tubular cells.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
HYPOKALEMIC METABOLIC TUBULOPATHY is an inherited renal tubular disorder. Patients with hypokalemic metabolic tubulopathy have often been called Bartter syndrome. Currently, patients with hypokalemic metabolic tubulopathy can generally be divided into three different clinical entities (1): neonatal Bartter syndrome (clinical signs may be observed before or immediately after birth and include marked polyhydramnios, premature delivery, massive polyuria, life-threatening episodes of dehydration, hypercalciuria, and nephrocalcinosis) (2); classic Bartter syndrome (typically, symptoms start during the first 2 yr of life and include polyuria and polydipsia, salt craving, fever, vomiting, dehydration, and failure to thrive; an important urinary finding is the presence of normal to high urinary calcium excretion); and Gitelman syndrome (patients do not usually have symptoms throughout infancy and the preschool years and are often diagnosed in adolescence or early adulthood). The symptoms are relatively mild. The outstanding laboratory findings are hypomagnesemia and hypocalciuria.

Advances in molecular genetics have clarified that these disorders are caused by renal tubular electrolyte transporter dysfunction. Neonatal Bartter syndrome may be linked to dysfunction of NKCC2 (Na-K-2Cl cotransporter) or ROMK (renal outer medullary K channel) (3, 4). Classic Bartter and Gitelman syndromes may be linked to dysfunction of ClC-Kb (Cl channel Kb) and NCCT (Na-Cl cotransporter), respectively (5, 6). However, a recent investigation showed that the phenotype-genotype correlations, especially in classic Bartter syndrome and Gitelman syndrome, are not as clear as the above classifications may suggest (7, 8). We report here two Japanese patients with clinically diagnosed Bartter syndrome. They showed hypocalciuria, which is generally considered a pathognomonic finding of NCCT dysfunction.

	Patients and Methods

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Case reports

Case 1. The 8-yr-old boy had been born at 38 wk gestation. His birth weight was 2535 g. At 4 months of age, he was brought to the pediatric office because of poor feeding and failure to thrive. The symptoms were so severe that he was referred to a hospital for further treatment and evaluation. He was found to have hypokalemia and metabolic alkalosis and was diagnosed as Bartter syndrome. Accordingly, oral potassium, sodium chloride, and potassium-sparing diuretics were commenced. His gross motor development was almost normal until late infancy. He could walk alone at 1 yr 4 months of age. However, he still showed a failure to thrive and exhibited salt craving and polydipsia with polyuria. At 4 yr of age, treatment with a prostaglandin synthesis inhibitor, indomethacin, was commenced. At 6 yr of age, mental retardation became prominent. The developmental quotient was 50. He entered elementary school but could not catch up because of his delayed growth and development. He rarely communicated with other people and was suspected to have a communication disorder. Therefore, he was referred to a pediatric psychiatric department. In addition, he suffered a right tibial and fibular bone fracture when he stumbled. He later suffered a right tibial and left fibular bone fracture without any prominent cause. At 7 yr of age, he showed prominent growth (height, –5.7 SD) and mental retardation. Serum sodium, chloride, and potassium levels were 130, 81, and 2.1 mmol/liter, respectively, under oral supplementation with potassium L-aspartate (4.3 mEq/kg·d). The urine calcium to creatinine molar ratio was 0.07 (normal, 0.2 to 0.6). His older sister showed similar symptoms. However, we could not analyze her genotype because she was living with her mother, and her father did not know where they lived (the mother and father are divorced). The father did not have any apparent clinical symptoms and refused to allow us to analyze his genotype.

Case 2. The patient is a 13-yr-old boy. He was born by vaginal delivery at 38 wk gestation. Birth weight was 2500 g. At 6 months of age, he was brought to the hospital because of fever. He then showed a marked failure to thrive (weight, 5250 g, –3.1 SD; length, 59.0 cm, –3.6 SD). On admission, he showed remarkable electrolyte disturbance. Serum sodium, chloride, and potassium were 123, 68, and 1.8 mmol/liter, respectively. Other laboratory findings were as follows: serum calcium, 4.1 mEq/liter; serum magnesium, 0.98 mmol/liter; and urinary sodium, 28 mmol/liter. Metabolic alkalosis was remarkable, with a plasma pH of 7.54, bicarbonate of 29.6 mmol/liter, base excess of 6.6 mmol/liter, and pCO2 of 34.9 mm Hg. Treatment with oral potassium supplement was started. His development was normal; however, his growth remained severely retarded. At 3 yr 6 months of age (height, 81 cm, –5.5 SD; weight, 9.2 kg, –3.5 SD), indomethacin was commenced after a diagnosis of Bartter syndrome. In addition, growth hormone therapy was begun at 5 yr 2 months of age to treat impaired growth hormone secretion. Since then, he has been showing catch-up growth. Now, at 13 yr of age, his height and weight are 151 cm (–1.2 SD) and 44.5 kg (–0.5 SD), respectively. His urinary calcium excretion is still remarkably low (urine calcium to creatinine molar ratio is 0.02) even though other laboratory findings are almost normal, under oral supplementation with potassium and indomethacin. Important clinical characteristics and representative biochemical data are shown in Table 1. Both of his parents did not have any apparent clinical symptoms.

Mutation analysis: sets of primer pairs for renal tubular electrolyte transporter genes

NKCC2 gene. Specific primer pairs that amplified the splice sites and the 26 exons of this gene were used. Primer pairs were also chosen to amplify two additional isoforms of exon 4.

ROMK gene. The gene encoding human ROMK produces five distinct transcripts (ROMK-1–5) by differential splicing of its five exons (9). All five transcripts share an exon 5 that encodes most of the ROMK protein. Seven sets of primer pairs were used to amplify the coding sequences for exons 1–5.

ClC-Kb gene. Primers that amplify the 19 exons of the ClC-Kb gene together with their splice sites were used.

NCCT gene. Twenty-six pairs of specific primers were used to amplify all 26 exons.

We screened these four genes by using primers that have been described previously (7).

PCR and direct sequencing

Genomic DNA was isolated from blood cells using a PUREGEN DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN). The exonic regions of each of the four transporters, including intron/exon boundaries, were amplified using specific primers by PCR. PCR was carried out using the following conditions: initial denaturation at 95 C for 10 min was followed by 35 cycles of denaturation at 95 C for 30 sec, annealing at 51 to approximately 72 C for 30 sec, and extension at 72 C for 1 min in 25 ml of reaction mixture containing 50 to approximately 100 ng genomic DNA, 1x PCR buffer (Applied Biosystems, Foster City, CA), 0.2 mM dNTPs, 0.5 mM each primer, and 0.5 U AmpliTaq DNA polymerase (Applied Biosystems) with a GeneAmp 9700 system (Applied Biosystems). The reaction was completed with a final elongation step at 72 C for 10 min. Finally, PCR products were purified with a QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The abundance and quality of DNA fragments were analyzed by electrophoresis on 1.2% agarose gel, followed by ethidium bromide staining and inspection under UV light. Direct sequencing of double-strand DNA fragments of exons of the four transporters was performed using internal sequencing primers with an ABI PRISM Bigdye Thermal Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and an ABI model 310 auto sequencer.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Sequence analysis of the four electrolyte transporter genes in patient 1 revealed two ClC-Kb allelic variants, which caused an amino acid substitution. We defined cDNA position 1 as the first base of the initiator methionine. One variant was a homozygous T to G substitution at cDNA position 80 in exon 1, which causes a Leu-to-Arg substitution at position 27 (L27R). The second variant was a heterozygous G to A substitution at cDNA position 1830 in exon 16, which introduces a preliminary stop codon at position 610 (W610X) (Fig. 1). There were no other mutations that led to an amino acid change in the other three transporter genes.

View larger version (44K): [in this window] [in a new window]   FIG. 1. Mutations in the ClC-Kb gene. A, In patients 1 and 2, the arrow shows the site of the heterozygous G to A substitution in exon 16. This substitution leads to a premature stop instead of tryptophan in one allele. B-1, In patient 2, the arrow shows the site of a heterozygous G to C substitution of the 3' splice site of intron 2. This mutation may result in aberrant mRNA splicing instead of an amino acid substitution in one allele.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
In the present study, we identified three ClC-Kb allelic variants, L27R, W610X, and a G to C substitution of the 3' splice site of intron 2, in two Japanese patients with hypokalemic metabolic alkalosis. Among them, L27R was not uncommon in the general population (40 of 160 alleles) in our previous study (7) and is considered an innocuous polymorphism (10). The W610X variant has not been reported previously, and both our patients shared this mutation. Even though further in vitro studies are needed to clarify how this mutation influences physiological function, we can offer some possible explanations for how W610X may influence physiological ClC-Kb function. W610X truncates the ClC-Kb protein by 78 amino acids at the carboxy-terminal end. ClC-Kb is made up of 687 amino acids and membrane-associated part of the protein composed of 17 -helices (11, 12). This means that this truncation of ClC-Kb protein loses over 10% of its amino acids. The splice site mutation, a G to C substitution of the 3' splice site of intron 2, may result in exon skipping or aberrant RNA splicing because exon-intron boundaries are strongly conserved splice-site consensus sequences. Because parents of patient 2 were heterozygotes for W610X and a G to C substitution of the 3' splice site of intron 2, respectively, and they had no apparent clinical symptoms, each of these mutations on one allele is not responsible for the phenotype in patient 2. It seems rational to consider that two mutations on separate alleles in patient 2 might be responsible for phenotype in patient 2. In the case of patient 1, we could not provide a reasonable answer to explain his phenotype. Based on our previous data (7), allele frequency of L27R was 0.25. This means frequency of homozygous L27R was 1.56% in general population. Homozygous L27R in concert with the heterozygous W610X may affect ClC-Kb function in patient 1.

Recent reports have suggested that the phenotype-genotype correlation is still confusing, especially in classic Bartter syndrome and Gitelman syndrome, despite advances in molecular genetics (13). Hypocalciuria seems to be a direct consequence in disorders of the distal convoluted tubule, as demonstrated in NCCT knockout mice (14), as well as in patients with long-term administration of thiazide diuretics, which inhibit NCCT (15). However, Konrad et al. (10) reported that three of 36 patients with clinically diagnosed classical Bartter syndrome with ClC-Kb mutation were hypocalciuric. Furthermore, Peters et al. (8) reported that hypocalciuria was not a rare symptom in patients with ClC-Kb mutation. Recently, Kobayashi et al. (16) demonstrated that ClC-Kb was also likely to be expressed in distal tubules and was physiologically related to NCCT. Taken together, these results suggest that malfunction of ClC-Kb may disturb the intracellular chloride concentration and cause secondary NCCT dysfunction.

Received April 26, 2004.

Accepted August 5, 2004.

	References

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