Endocr. J./ T.TAJIMA et al.: MUTATIONS OF THE CLCNKB doi:10.1507/endocrj.K06-034

ORIGINAL

Molecular analysis of the CLCNKB gene in Japanese patients with classic

TOSHIHIRO TAJIMA, MITSURU NAWATE*, YUTAKA TAKAHASHI*, YUMIKO MIZOGUCHI**, # SHIGETAKA SUGIHARA**, MASAAKI YOSHIMOTO***, MUTSUMI MURAKAMI , MASANORI ## ## ### ADACHI , KATSUHIKO TACHIBANA , HIROSHI MOCHIZUKI , KENJI FUJIEDA♭

Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, 060-8638, Japan * Department of Pediatrics, Konan Hospital, Sapporo, 062-093, Japan ** Department of Pediatrics, Tokyo Women’s Medical University, Daini Hospital, Tokyo, 16-8567, Japan *** Yoshimoto Pediatric Clinic, Nagasaki, 852-8154, Japan # Department of Pediatrics, Nihon Medical School, Tokyo, 113-8603, Japan ## Department of Endocrinology and Metabolism, Kanagawa Children’s Medical Center, Kanagawa, 232-8555, Japan ### Department of Endocrinology and Metabolism, Saitama Children’s Medical Center, Saitama, 339-8551, Japan ♭Department of Pediatrics, Asahikawa Medical College, Asahikawa, 078-8510, Japan

Received February 15, 2006; Accepted June 2, 2006; Released online August 11, 2006

Correspondence to: Toshihiro TAJIMA, MD, PhD, Department of Pediatrics, Hokkaido University School of Medicine, Kita-ku, N15, W7, Sapporo, 060-8638, Japan

Abstract. Deletions or mutations in the gene encoding the basolateral CLC-Kb (CLCNKB) cause classic Bartter syndrome (MIM 602023), which is characterized by hypokalemic metabolic alkalosis, hyperreninemic hyperaldosteronism and hypercalciura. These patients are usually diagnosed during infancy or childhood due to failure to thrive and growth retardation. The purpose of this study was to investigate the underlying mutations in Japanese patients with classic Bartter syndrome. Seven Japanese patients from seven different families diagnosed as having classic Bartter syndrome were studied. Analysis of CLCNKB demonstrated a large deletion in two patients, a partial deletion in one patient and two mutations (Δ L130 in exon 4 and W610X in exon 16) in the remaining four patients. Δ L130 is a novel mutation, but W610X was previously reported in three unrelated Japanese patients. Six out of the seven patients were diagnosed due to typical characteristics of classic Bartter syndrome such as failure to thrive and poor weight gain however, one patient was asymptomatic with mild hypokalemia. In conclusion, we identified a novel mutation of the CLCNKB gene, Δ L130. We did not determine whether the W610X mutation in our patients was from a common ancestor or if this mutation is frequent in Japan.

Key words; CLCNKB, Hypokalemia, Novel mutation, W610X

HEREDITARY salt-losing renal tubular diseases with secondary hypokalemia are traditionally categorized into ‘Bartter syndrome’, as they share several clinical and biochemical findings such as renal salt loss, hypokalemic hypochloremic metabolic alkalosis, and normal blood pressure despite hyperreninemic hyperaldosteronism [1-4]. Based on clinical manifestations, additional symptoms and the biochemical profile, especially with respect to calcium and magnesium handling, at least four different types of disease have been proposed: (1) the classic Bartter syndrome, (2) the hypomagnesemic hypocalciuric Gitelman syndrome, (3) antenatal Bartter syndrome, and (4) antenatal Bartter syndrome with sensorineural deafness. The latter two syndromes are the most severe forms, in which clinical symptoms of massive polyuria, polyhydramnios, severe life-threatening salt wasting, hypokalemic metabolic alkalosis, hypercalciuria and markedly medullary nephrocalcinosis occur during the prenatal and neonatal periods [2-5].

1 Endocr. J./ T.TAJIMA et al.: MUTATIONS OF THE CLCNKB GENE doi:10.1507/endocrj.K06-034

In the last two decades, the molecular genetics of inherited salt-losing tubular disorders with secondary hypokalemia has become much clearer, such that clinical differentiations have been classified by the underlying molecular defects. To date, inactivating mutations in six different encoding renal membrane involved in electrolyte reabsorption have been detected. In antenatal patients, mutations occur in either the furosemide sensitive sodium-potassium-chloride cotransporter (NKCC2) or in the ROMK. Antenatal patients with sensorineural deafness have mutations in the β-subunits of the chloride channels ClC-Kb and ClC-Ka (barttin). The classic Bartter syndrome is caused by genetic defects in CLCNKB, and Gitelman syndrome is caused by mutations in the thiazide-sensitive sodium-chloride cotransporter NCCT. Finally, one unique patient’s disease was caused by a CLCNKA mutation coupled with CLCNKB deletion [6-12]. The classic Bartter syndrome caused by CLCNKB defects usually presents during infancy or early childhood. The symptoms of these patients include salt-losing hypokalemia, polyuria, polydipsia, failure to thrive, and growth retardation, which best fit with the original description given by Bartter et al. [1]. However, there is phenotypic variability of CLCNKB defects ranging from severe symptoms like antenatal Bartter syndrome to those almost identical to Gitelman syndrome [11,13-16]. CLCNKB encodes the ClC-Kb, which mediates chloride efflux from tubular epithelial cells to the interstitium along the thick ascending limb of the loop of Henle (TAL) and the distal convoluted tubule (DCT) [8, 11]. At present a large deletion, a partial deletion, splicing, missense and nonsense mutations of CLCNKB have been reported [8, 11, 13-18]. In this study, we analyzed the molecular basis of seven Japanese patients with classic Bartter syndrome.

Methods Patients Seven Japanese patients from seven unrelated families were studied. Parents of children gave their informed consent to participate in the study. This study was approved by the ethical committee of the Hokkaido University School of Medicine. Diagnosis of classic Bartter syndrome was based on the clinical and biochemical findings. These findings at diagnosis are summarized in Table 1.

DNA amplification and sequence analysis Genomic DNA was extracted from peripheral leukocytes. To detect deletions of the CLCNKB gene, long-range PCR for exons 1 to 2, 6 to 9, and 17 to 19 were performed according to previous reports using LA Taq polymerase (Takara Co, Tokyo, Japan) [8,11]. Each exon of CLCNKB was specifically amplified by PCR as mentioned by Konrad et al. (11). All primers for PCR of CLCNKB were identical to previous studies and we sequenced all exons and exon-intron boundaries [8, 11]. The PCR was performed in a Perkin-Elmer Gene Amp PCR System 2400 thermal cycler (PE Applied Biosystems, Foster City, CA, USA). After amplification, the PCR products were purified from low melting agarose gel. The purified products were sequenced directly with an ABI PRISM Dye Terminator Cycle Sequencing Kit and an ABI 373A automated fluorescent sequencer (PE Applied Biosystems, Foster City, CA).

Results Clinical and biochemical findings The clinical and laboratory findings of the seven patients are summarized in Table 1. Six patients showed growth retardation and failure to thrive, which are typical symptoms

2 Endocr. J./ T.TAJIMA et al.: MUTATIONS OF THE CLCNKB GENE doi:10.1507/endocrj.K06-034 of the classic Bartter syndrome. These patients also had hypokalemic alkalosis and hyperreninemic hyperaldosteronism. The urinary calcium /creatinine ratio in these patients was high (Table 1). We determined that the serum magnesium levels of patients 2, 3, 4, and 7, and the levels of these patients were within normal range. Patient 7 had not shown any symptoms until this study was performed. He was diagnosed at 2 years of age when routine blood examination revealed possible hypokalemia (3.1 mEq/L). Repeated measurements confirmed hypokalemia. His aldosterone level (45 pg/ml, normal range 35-240 pg/ml) was within normal range; however, his plasma renin activity (PRA) was mildly elevated (10.5 ng/ml/hr, normal range, 0.3-5.4 ng/ml/hr). His urinary calcium /creatinine ratio (0.35 mg/mg) was slightly higher than normal. Thus, he was suspected to have a mild form of the classic Bartter syndrome.

Molecular analysis Three patients had deletion mutations in the CLCNKB gene. PCR analysis of the CLCNKB gene showed a large deletion in patients 1 and 2. Long-range PCR and PCR for each exon of CLCNKB did not amplify any part of the gene, indicating a homozygous deletion, as reported previously [8, 11, 13]. In patient 7, long range PCR for exons 1 to 2 did not amplify CLCNKB; however, long range PCR for exons 6 to 9 and 17 to 19 successfully produced the expected bands (data not shown). In addition, specific amplification for exons 1 and 2 did not produce the expected bands, even though the other exons (exons 3 to 19) were successfully amplified. These results indicate partial deletion of the 5’ portion of the CLCNKB gene [8,11]. In the remaining four patients, one amino acid deletion and one nonsense mutation were identified. Patient 3 had a heterozygous deletion mutation (ΔL130) in exon 4 (Fig.1). Family analysis demonstrated that his mother also had this mutation in the heterozygous state; however, she did not manifest any symptoms. The father of patient 3 was also asymptomatic, and sequencing of the paternal gene did not identify any mutations in CLCNKB. The W610X mutation in exon 16 was identified in Patients 4, 5, and 6. In patient 4, this mutation was homozygous (Fig. 2). Patients 5 and 6 had this mutation in the heterozygous state (Fig. 2); however, we did not identify any other mutations. Patients 4, 5 and 6 were unrelated.

Discussion We analyzed seven patients with classic Bartter syndrome and identified genetic alternations of CLCNKB in all patients. Among them, one mutation was novel (ΔL130). The functional consequence of ΔL130 was not determined however, ΔL 130 was not identified in 100 control Japanese subjects. Furthermore, this leucine residue at position 130 is highly conserved between species and within the chloride channel gene family [8, 11, 19-21]. In addition, a P124L missense mutation located in the identical domain as L130 was found in three Turkish patients and one Italian patient with classic Bartter syndrome [8]. Thus, this domain is functionally important and Δ130L may impair the function of CLC-Kb. The W610X mutation was identified in three unrelated patients. This premature stop codon is expected to produce the truncated CLC-Kb, resulting in the loss of function. Previous studies have reported the W610X mutation in three other Japanese patients [17,18]. Thus, six out of ten Japanese patients analyzed had the W610X mutation. As this mutation was not observed in other ethnic origins, it is likely to have arisen from a common ancestor in Japan. In three patients (patients 3, 4 and 6), we identified only one mutation on one allele, although we sequenced all exons and exon-intron boundaries. Thus, mutations and/or

3 Endocr. J./ T.TAJIMA et al.: MUTATIONS OF THE CLCNKB GENE doi:10.1507/endocrj.K06-034 partial deletions may have been missed in the other allele. The heterozygosity found in our study is likely due to the limitation of the PCR-based method to identify small heterozygous deletions. Alternatively, it may be possible that any other genetic defect of these patients is located 5’ upstream, 3’ downstream and/or within intronic regions. Therefore, further studies are warranted. A large deletion of the CLCNKB gene in two patients and a partial deletion of the 5’ region of the CLCNKB gene in one patient were also identified. As described previously, deletions and partial loss of the CLCNKB gene are frequent. These deletions could have resulted from nonhomologous recombination between the CLCNKB and CLCNKA genes, which are almost identical in sequence and are very closely located on the 1 [8, 11]. As mentioned, varying degrees of disease severity ranging from asymptomatic to severe polyuria and salt loss during the prenatal and neonatal period have been reported [11, 13-16]. In accordance with previous reports, one patient was asymptomatic and was diagnosed only when hypokalemia was discovered during a routine blood examination. Biochemical abnormalities were also very mild in this patient. It has been speculated that the mutant genotypes of CLCNKB could explain the degree of clinical severity; however, two studies could not clarify the genotype-phenotype correlation [11,13,15]. One plausible explanation of the phenotypic variability is due to the broad expression pattern of ClC-Kb [8, 11]. Alternatively, other routes could compensate for Cl- transport such as KCL cotransporters, transmembrane regulators or the voltage-gated Cl- channel (CLC5) [22, 23]. These compensatory mechanisms may modify the degree of impaired Cl- transport imposed by CLC-Kb defects and thus influence the phenotypes of the disease.

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10. Birkenhager R, Otto E, Schurmann MJ, Vollmer M, Vollmer M, Ruf EM, Maier-Lutz I, Beekmann F, Fekete A, Omran H, Feldmann D, Milford DV, Jeck N, Konrad M, Landau D, Knoers NV, Antignac C, Sundbrak R, Kispert A, Hildebrandt F (2001) Mutation in BSND causes Bartter syndrome with sensorineural deafness and kidney failure. Nat Genet 29:310-314. 11. Konrad M, Vollmer M, Lemmink HH, van den Heuvel LP, Jeck N, Vargas-Poussou R, Lakings A, Ruf R, Deschenes G, Antignac C, Guay-Woodford L, Knoers NV, Seyberth HW, Feldmann D, Hildebrandt F (2000) Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome. J Am Soc Nephrol 11:1449-1459. 12. Schlingmann KP, Konrad M, Jeck N, Waldegger P, Reinalter SC, Holder M, Seyberth HW, Waldegger S (2004) Salt wasting and deafness resulting from mutations in two chloride channels. N Engl J Med 350:1314-1319. 13. Jeck N, Konrad M, Peters M, Weber S, Bonzel KE, Seyberth HW (2000) Mutations in the chloride channel gene, CLCNKB, leading to a mixed Bartter-Gitelman phenotype Pediatr Res 48:754-758. 14. Schurman SJ, Perlman SA, Sutphen R, Campos A, Garin EH, Cruz DN, Shoemaker LR (2001) Genotype/phenotype observations in African Americans with Bartter syndrome. J Pediatr 139:105-110 15. Peters M, Jeck N, Reinalter S, Leonhardt A, Tonshoff B, Klaus G, Konrad M, Seyberth HW (2002) Clinical presentation of genetically defined patients with hypokalemic salt-losing tubulopathies. Am J Med 112:183-190 16. Zelikovic I, Szargel R, Hawash A, Labay V, Hatib I, Cohen N, Nakhoul F (2003) A novel mutation in the chloride channel gene, CLCNKB, as a cause of Gitelman and Bartter syndromes. Kidney Int 63:24-32. 17. Fukuyama S, Hiramatsu M, Akagi M, Higa M, Ohta T (2004) Novel mutations of the chloride channel kb gene in two Japanese patients clinically diagnosed as Bartter syndrome with hypocalciuria. J Clin Endocrinol Metab 89:5847-5850. 18. Watanabe T, Tajima T (2005) Renal cysts and nephrocalcinosis in a patient with Bartter syndrome type III. Pediatr Nephrol 20:676-678. 19. Fisher SE, van Bakel I, Lloyd SE, Pearce SH, Thakker RV, Craig IW (1995) Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis. Genomics 29:598-606. 20. Brandt S, Jentsch TJ (1995) ClC-6 and ClC-7 are two novel broadly expressed members of the CLC chloride channel family. FEBS Lett 377:15-20. 21. Mount DB, Mercado A, Song L, Xu J, George AL, Delpire E, Gamba G (2000) Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family. J Biol Chem 274:16355-16362. 22. Devuyst O, Burrow CR, Schwiebert EM, Guggino WB, Wilson PD (1996) Developmental regulation of CFTR expression during human nephrogenesis. Am J Physiol 271:F723-735 23. Luyckx VA, Goda FO, Mount DB, Nishio T, Hall A, Hebert SC, Hammond TG, Yu A (1998) Intrarenal and subcellular localization of rat CLC5. Am J Physiol 275:F761-769.

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Fig. 1. In one patient, a novel deletion mutation caused Bartter syndrome. Sequence analysis of patient 3 demonstrated a 3 base inframe deletion (ΔL 130) (arrow). This mutation was heterozygous. Note the double nucleotides after the mutation site. WT indicates the sequence of the wild-type gene.

Fig. 2. In three patients, a nonsense mutation caused Bartter syndrome. Sequence analysis demonstrated a G to A change. Arrows indicate the mutation. This base change introduced a premature stop codon (TGG to TGA). Patient 4 was homozygous for this mutation, and Patients 5 and 6 were heterozygous for this mutation. WT indicates the sequence of the wild-type gene.

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