A Novel Mutation in the Chloride Channel Gene, CLCNKB, As a Cause of Gitelman and Bartter Syndromes

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Kidney International, Vol. 63 (2003), pp. 24–32

GENETIC DISORDERS – DEVELOPMENT

A novel mutation in the chloride channel gene, CLCNKB, as a cause of Gitelman and Bartter syndromes

ISRAEL ZELIKOVIC,RAYMONDE SZARGEL,ALI HAWASH,VALENTINA LABAY,1 IHAB HATIB, NADINE COHEN,2 and FARID NAKHOUL

Pediatric Nephrology Unit and Department of Nephrology, Rambam Medical Center, and Department of Molecular Genetics, Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel

Ϫ A novel mutation in the chloride channel gene, CLCNKB,asa linkage to the Cl channel gene, CLCNKB, on chromosome cause of Gitelman and Bartter syndromes. 1p36. Mutation analysis by direct sequencing revealed a novel Background. Gitelman syndrome (GS) and Bartter syn- homozygous missense mutation, arginine 438 to histidine drome (BS) are hereditary hypokalemic tubulopathies with (R438H), in exon 13 of CLCNKB in all patients. A restriction distinct phenotypic features. GS has been considered a geneti- fragment length polymorphism (RFLP) analysis has been de- cally homogeneous disorder caused by mutation in the gene veloped to aid in genotyping of family members. encoding the NaCl cotransporter (TSC) of the distal convo- Conclusions. Our findings demonstrate intrafamilial hetero- luted tubule. In contrast, BS is caused by mutations in the geneity, namely the presence of GS and CBS phenotypes, in genes encoding either the Na-K-2Cl cotransporter (NKCC2), a kindred with the CLCNKB R438H mutation. We conclude the Kϩ channel (ROMK) or the ClϪ channel (ClC-Kb) of the that GS can be caused by a mutation in a gene other than thick ascending limb. The purpose of this study was to examine SLC12A3. The exact role of the CLCNKB R438H mutation the clinical, biochemical and genetic characteristics of a very in the pathogenesis of the electrolyte and mineral abnormalities large inbred Bedouin kindred in Northern Israel with heredi- in GS and CBS remains to be established. tary hypokalemic tubulopathy. Methods. Twelve family members affected with hypokalemic tubulopathy, as well as 26 close relatives were clinically and biochemically evaluated. All study participants underwent Bartter syndrome is a group of closely related heredi- genetic linkage analysis. Mutation analysis was performed in tary tubulopathies. All variants of the syndrome share affected individuals. several clinical characteristics including renal salt wasting, Results. Evaluation of affected family members (age range hypokalemic metabolic alkalosis, hyperreninemic hyper- 3 to 36 years) revealed phenotypic features of both GS and classic Bartter syndrome (CBS). Features typical of GS in- aldosteronism with normal blood pressure and hyperpla- cluded late age of presentation (Ͼ15 years) in 7 patients (58%), sia of the juxtaglomerular apparatus [1–3]. The syndrome normal growth in 9 (75%), hypomagnesemia (SMg Ͻ0.7mmol/L) has been classified into three distinct phenotypes. First, Ͼ in 5 (42%), hypermagnesiuria (FEMg 5%) in 6 (50%) and hy- antenatal Bartter syndrome, also known as hyperprosta- Ͻ pocalciuria (urinary calcium/creatinine mmol/mmol 0.15) in glandin E syndrome, is the most severe form of the dis- 5 (42%). Features typical of CBS included early age of presentation (Ͻ1 year) in 3 (25%), polyuria/dehydration in 4 (33%), ease. It is characterized by polyhydramnios, premature growth retardation in 3 (25%), hypercalciuria (urinary calcium/ birth, life threatening episodes of salt and water loss in creatinine mmol/mmol Ͼ0.55) in 4 (33%) and nephrolithiasis the neonatal period, hypokalemic alkalosis and failure in 1 (8%). Linkage analysis in affected patients excluded the to thrive, as well as hypercalciuria and early onset nephro- TSC gene, SLC12A3, as the mutated gene, but demonstrated calcinosis [1, 2]. Second, classic Bartter syndrome occurs in infancy or early childhood. It is characterized by marked 1 Current address: National Institute on Deafness and other Communi- salt wasting and hypokalemia leading to polyuria, poly- cation Disorders, National Institute of Health, 5 Research Court, Room dipsia, volume contraction, muscle weakness, and growth 2A-02, Rockville, MD 20850, USA. 2 Current address: Johnson and Johnson Pharmaceutical Research and retardation. Hypercalciuria and nephrocalcinosis may Development, 1000 Route 202, P.O. Box 300, Raritan, NJ 08869, USA. occur [2, 3]. Finally, Gitelman syndrome is characterized by a milder clinical presentation in older children or adults Key words: hereditary hypokalemic tubulopathy, chloride channels, magnesium deficiency, phenotype, linkage (genetics), sequence analy- [4]. Patients may be asymptomatic and present with tran- sis, DNA, Bedouin kindred study. sient muscle weakness, abdominal pain, symptoms of neuromuscular irritability, or unexplained hypokalemia. Received for publication May 13, 2002 and in revised form July 18, 2002 Hypocalciuria and hypomagnesemia are typical [3]. Accepted for publication August 28, 2002 The genetic variants of Bartter syndrome that have been  2003 by the International Society of Nephrology identified include Bartter syndrome types I, II, and III,

24 Zelikovic et al: Mutation in Gitelman and Bartter syndromes 25 which are caused by mutations in the genes encoding ples for biochemical analyses were collected after appro- the luminal Naϩ-Kϩ-2ClϪ cotransporter, NKCC2 [5], the priate informed consent was obtained from the subjects luminal Kϩ channel, ROMK [6], and the basolateral ClϪ or their guardian. Serum levels of potassium, creatinine, channel, ClC-Kb [7], respectively, of the thick ascending calcium and magnesium as well as plasma bicarbonate limb of the loop of Henle (TAL). In contrast, Gitelman level were analyzed using standard laboratory automated syndrome, which has been considered a genetically ho- techniques. Urine was collected for analysis of calcium, mogenous disorder, is caused by mutations in the gene magnesium and creatinine levels. Calcium excretion was encoding the thiazide-sensitive NaCl cotransporter, TSC, expressed as the urinary calcium/creatinine ratio and mag- of the distal convoluted tubule (DCT) [8]. nesium excretion was expressed as fractional excretion With respect to genotype-phenotype correlation, it of magnesium. has been widely accepted that patients with the NKCC2 [5, 9, 10] or ROMK [6, 11, 12] defect usually have the se- Genetic linkage analysis vere antenatal form, those with the ClC-Kb defect have Genomic DNA was extracted from peripheral blood the classic form [7], and the TSC defect leads to Gitelman lymphocytes of family members by using standard meth- syndrome [8, 13]. ods [19]. Linkage analysis was performed using 4-6 dinu- Recent data, however, have suggested that the geno- cleotide repeat (CA) markers from the Genethon micro- type-phenotype correlation is not so clear-cut and that satellite panel [20] tightly linked to each of the following phenotypic overlap may occur. Konrad et al [14] and Jeck three genetic loci: Naϩ-Kϩ-2ClϪ cotransporter gene (SLC- et al [15] reported that mutations in the ClC-Kb gene, 12A1), thiazide-sensitive NaCl cotransporter gene (SLC- CLCNKB, also can cause phenotypes that overlap with 12A3) and voltage-gated ClϪ channel gene (CLCNKB). either antenatal Bartter syndrome or Gitelman syndrome. The following markers were used: D15S132, D15S1017, Recently, an additional variant of antenatal Bartter syn- D15S978, D15S126, D15S121, D15S209 (for SLC12A1), drome associated with sensorineural deafness and renal D16S408, D16S3140, D16S3071, D16S494 (for SLC12A3) failure has been described [16]. This variant is caused by and D1S2672, D1S507, D1S2697, D1S436 (for CLCNKB). mutations in the BSND gene, which encodes the integral Genotyping of the markers was performed by use of a membrane protein barttin [17], a ␤-subunit for voltage- non-radioactive–labeling procedure with the enhanced gated (CLC) ClϪ channels [18]. chemiluminescence (ECL) system (Amersham, Braun- The purpose of the current study was to shed addi- schweig, Germany), on the basis of a horseradish peroxi- tional light upon this phenotypic heterogeneity by exam- dase-mediated chemiluminescent reaction [21]. ining the clinical, biochemical and genetic characteristics of a very large inbred Bedouin kindred in Northern Israel Mutation analysis by direct sequencing with hereditary hypokalemic tubulopathy, in which a Nineteen pairs of oligonucleotide primers were gener- wide spectrum of phenotypic features in affected patients ated to amplify by polymerase chain reaction (PCR) all has been noted. 19 exons of the CLCNKB gene according to data obtained from Simon et al [7]. PCR-amplified coding regions as well as exon-intron boundaries were sequenced. METHODS PCR conditions for genomic amplification were as fol- Patients lows: Denaturation at 94ЊC for two minutes; 35 subse- A large inbred Bedouin family residing in two close quent amplifications cycles performed at 94ЊC for 30 sec- villages in the lower Galilee was investigated. Twelve onds, 55ЊC for 30 seconds and 72ЊC for seven minutes. members of the family were identified as having hypoka- Amplified DNA fragments were analyzed by electropho- lemic tubulopathy. The pedigree of the kindred is shown resis on 2% agarose gels and purified using the QIAquick in Figure 1. The family has lived in the same area for gel extraction kit. Then, purified PCR products were more than two centuries. The pattern of inheritance of sequenced using capillary electrophoresis on an ABI the disease in the family is compatible with an autosomal PRISM 310 automated sequencer (Perkin Elmer, War- recessive trait. Twelve family members affected with hy- rington, UK) using the Big Dye terminator cycle se- pokalemic tubulopathy and 26 close relatives were in- quencing kit (Perkin Elmer). Mutation analysis was per- cluded in this study. At the time of enrollment, therapy formed in ten of the twelve affected family members. of patients included various combinations of potassium supplements, amiloride and spironolactone. No patient Restriction fragment length polymorphism analysis received magnesium supplementation. Restriction fragment length polymorphism (RFLP) analysis of exon 13 of CLCNKB was performed. PCR- Phenotype analysis amplified DNA fragments containing the locus mutated Clinical data were obtained from the patients, their in this family were digested with the restriction enzyme parents and their care providers. Blood and urine sam- Kas I and electrophoresed on a 2% agarose gel. Fig. 1. Extended pedigree of a Bedouin family with hypokalemic tubulopathy. Filled squares (males) and circles (females) indicate affected individuals. Deceased individuals are indicated by a diagonal line. The autosomal recessive mode of inheritance is evident. Zelikovic et al: Mutation in Gitelman and Bartter syndromes 27

Table 1. Clinical features in affected patients Age Age at presentation History of Presence of Patient/pedigree no. years Growth polyuria/dehydration nephrolithiasis MZ/7 21 17 Normal No No RZ/9 28 20 Normal No No YZ/27 21 20 Normal No No NZ/6 36 24 Retarded No No MZ/10 32 20 Retarded No No YZ/1 31 25 Normal Yes Yes MZ/2 23 10 Normal Yes No MZ/12 10 4 Normal Yes No IZ/3 3 0.5 Retarded Yes No MZ/5 7 0.3 Normal No No NZ/13 3 0.1 Normal No No BZ/14 31 29 Normal No No

Table 2. Laboratory ﬁndings in affected patients

ϩ Ϫ ϩϩ Serum K Plasma HCO3 Serum Mg FEMg Urinary Ca/Cr mEq/L mEq/L mmol/L % mmol/mmol Patient/pedigree no. (nl 3.5–5.3) (nl 22–26) (nl 0.7–1.2) (nl Ͻ5%) (nl 0.15–0.55) MZ/7 3.1 27 0.65 2.6 0.08 RZ/9 2.5 30 0.95 8.9 0.64 YZ/27 2.3 32 0.85 4.9 0.34 NZ/6 2.8 30 0.6 5.3 0.05 MZ/10 3.0 28 0.8 2.5 0.17 YZ/1 2.9 35 0.6 8.8 0.14 MZ/2 2.7 33 0.6 4.3 0.11 MZ/12 3.1 27 0.9 6.7 0.62 IZ/3 3.0 28 0.8 9.0 1.70 MZ/5 2.8 32 0.75 3.5 0.11 NZ/13 2.9 27 0.8 7.5 0.84 BZ/14 3.4 35 0.65 4.1 0.22

FEMg is fractional excretion of magnesium.

RESULTS Table 3. Summary of phenotypic features in affected patients Clinical and biochemical characteristics Number of Feature patients % Tables 1 and 2 summarize the clinical features and Features typical of Gitelman syndrome laboratory findings in the 12 affected family members. Late age of presentation (Ͼ 15 years) 7 58 Age at enrollment in the study ranged from 3 years to Normal growth 9 75 Յ 36 years (median 21 years). Age at initial presentation Hypomagnesemia (SMg 0.7 mmol/L) 5 42 Ͼ ranged from one month to 29 years (median 17 years). Hypermagnesiuria (FEMg 5%) 6 50 Hypocalciuria [urinary Ca/Cr There were seven males and five females. All patients (mmol/mmol) Ͻ 0.15] 5 42 had normal blood pressure and normal glomerular fil- Features typical of classic Bartter syndrome tration rate. No patient had diarrhea during the study Early age of presentation (Ͻ1 year) 3 25 Polyuria/dehydration 4 33 period. The clinical and biochemical evaluation revealed Growth retardation 3 25 phenotypic features of both Gitelman syndrome (GS) Hypercalciuria [urinary Ca/Cr and classic Bartter syndrome (CBS; Table 3). Features (mmol/mmol) Ͼ 0.55] 4 33 typical of GS included late age at presentation (Ͼ15 Nephrolithiasis 1 8 years) in seven patients, normal growth in nine, hypo- Abbreviations are: SMg, serum magnesium; FEMg, fractional excretion of magnesium; Ca/Cr, calcium, creatinine. magnesemia (SMg Ͻ0.7mmol/L) in five, hypermagnesiuria (FEMg Ͼ5%) in six and hypocalciuria (calcium/creatinine mmol/mmol Ͻ0.15) in five. Features typical of CBS included early age of presentation (Ͻ1 year) in three, his- calciuria (Table 2), typical findings in GS, four had con- tory of polyuria and episodes of dehydration in four, comitant hypomagnesemia and hypocalciuria. However, growth retardation in three, hypercalciuria (urinary cal- generally, the group of affected patients could not be cium/creatinine mmol/mmol Ͼ0.55) in four and nephroli- subdivided into the “GS group” and the “CBS group,” thiasis in one patient. because the distinctive clinical/biochemical features of Of the six patients with hypomagnesemia and/or hypo- either GS or CBS did not segregate in the same patient. 28 Zelikovic et al: Mutation in Gitelman and Bartter syndromes

The same patient could have features of both GS and CBS. Nevertheless, ﬁve of the affected patients (pedigree numbers 1, 3, 6, 9 and 10) have been generally characterized by a more severe form of the disease including hypokalemia more resistant to therapy, frequent muscle symptoms, frequent hospitalizations, growth retardation, as well as polyuria and recurrent episodes of dehydration. There were no known cases with features of antenatal Bartter syndrome (such as polyhydraoannios, premature birth, manifestations in the neonatal period) and no reported cases of unexpected mortality in this family.

Linkage analysis Linkage analysis using the appropriate microsatellite markers (Methods section) ﬂanking the loci of the genes encoding the Naϩ-Kϩ-2ClϪ cotransporter (SLC12A1) and the thiazide-sensitive NaCl cotransporter (SLC12A3) did not reveal regions of homozygosity at these loci (data not shown). Figure 2 shows the results of haplotype analysis using microsatellite markers linked to the CLCNKB locus on chromosome 1p36. For all affected individuals in the six subfamilies of this large kindred, regions of homozygosity by descent were observed for this genomic region, which was then further analyzed.

Mutation analysis The linkage analysis data prompted a search for mutations in the CLCNKB gene. Mutation analysis revealed a novel homozygous missense mutation, guanine to adenine in exon 13 of CLCNKB (Fig. 3). The mutation substitutes arginine with histidine (R438H) at amino acid 438 of the channel protein. All ten affected individuals tested were found to carry the same mutation, homozygous by decent from their related heterozygous parents.

RFLP analysis Restriction fragment length polymorphism analysis of PCR-ampliﬁed fragments in exon 13 of CLCNKB containing the mutated locus in this family was performed. The R438H mutation in the variant allele in this family results in the loss of Kas I restriction enzyme site. As shown in Figure 4, the presence of the R438H mutation could be detected by RFLP analysis.

Fig. 2. Linkage analysis in six subfamilies of the large kindred with hypokalemic tubulopathy. Filled squares (males) and circles (females) indicate affected individuals. Each individual within the kindred is num- bered below the symbol. Haplotype analysis using microsatellite markers (shown on the left side) linked to the CLCNKB locus on chromosome 1p36 demonstrated cosegregation of the phenotype with the CLCNKB locus. Regions of homozygosity are framed. Zelikovic et al: Mutation in Gitelman and Bartter syndromes 29

Fig. 4. Restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction-ampliﬁed fragment in exon 13 of CLCNKB containing the locus mutated in this family. The numbers correspond to the number of the individual within the kindred (see Figs. 1 and 2). The cleavage site of the restriction enzyme Kas I in the wild-type 773 bp fragment is eliminated by the R438H mutation. Healthy individuals (wild-type) have two fast migrating bands, affected patients, homozy- gotes for the mutation (Mutant), have a single, slower migrating band and heterozygotes for the mutation have a three band pattern: two, fast migrating bands and one, slow migrating band.

Gitelman syndrome and classic Bartter syndrome can be distinguished by their characteristic phenotypic features, which include late age, benign course and presence of hypomagnesemia and hypocalciuria in the former and early onset, polyuria, volume contraction, failure to thrive and hypercalciuria in the latter [1–3]. Mutations in the thiazide-sensitive NaCl cotransporter gene, SLC12A3, on chromosome 16 have been found in nearly all GS patients studied [8, 13, 22]. On the other hand, in most patients with CBS, the molecular defect has been identified as a mutation in the voltage-gated ClϪ channel gene, CLCNKB, on chromosome 3 [7, 15]. The clinical and biochemical evaluation of affected members of the family reported here has revealed phenotypic features of both GS and CBS. An additional in- teresting characteristic of this family was the fact that, generally, the features of either GS or CBS did not segre- Fig. 3. Mutation analysis by direct sequencing in family members affected with hypokalemic tubulopathy. The sequence in the mutation gate in the same patient. Phenotypic features of both GS area for healthy individuals (A), heterozygotes (B) and affected ho- and CBS could be found in the same patient. In contrast to mozygotes (C) is listed below the histograms. The analysis reveals a the observed phenotypic heterogeneity within the family, novel homozygous missense mutation, guanine to adenine, in exon 13 of CLCNKB. The mutation substitutes arginine with histidine (R438H) all affected family members were found to have the same at amino acid 438 of the channel protein. genetic defect, a missense R438H mutation in exon 13 of the voltage-gated ClϪ channel gene, CLCNKB. Recent studies have provided evidence for phenotypic DISCUSSION overlap between the various forms of Bartter syndrome This study describes the clinical, biochemical and ge- including CBS and GS. Bettinelli et al, who studied 34 netic characteristics of the largest consanguineous kin- children with primary hypokalemic tubulopathy from 15 dred affected with hereditary hypokalemic tubulopathy centers in Central Europe, reported failure to thrive in reported to date. This study not only provides evidence 20% of the patients with GS phenotype and found hypo- that the genotype-phenotype correlation in Bartter/Git- magnesemia in one third of the children with BS pheno- elman syndrome is blurred, but also provides a unique type [23]. Thurman reported the concomitant occurrence opportunity to demonstrate the phenotypic variability of of Gitelman and Bartter syndromes in the same family this syndrome in members of the same family bearing the [24]. The author described two siblings presenting with same genetic defect. normotensive hypokalemic alkalosis. One child had hy- 30 Zelikovic et al: Mutation in Gitelman and Bartter syndromes pomagnesemia and hypocalciuria whereas the other had curs primarily via the luminal, thiazide-sensitive NaCl normal serum magnesium and urinary calcium excretion. cotransporter (TSC) [3, 25, 26]. Chloride exit to blood These two reports, however, did not include a genetic is mediated via basolateral ClϪ channels. The main path- analysis of affected patients. Simon et al, in their first way of ClϪ exit from cell to blood in the distal tubule ap- report on CLCNKB mutation in 17 kindreds with Bartter pears to be ClC-Kb channels, which are known to be syndrome type III, noted the marked variation in severity expressed at the basolateral membrane of the TAL and of clinical features of affected patients ranging from pro- the DCT [27–29]. Hence, a defect in the activity of found hypokalemia and severe volume depletion to mild ClC-Kb is expected to cause a reduction in NaCl reab- disease presenting at the second decade of life [7]. Kon- sorption in these tubule segments. Nevertheless, alterna- rad and coworkers, who screened children with Bartter’s tive routes for ClϪ transport such as KCl cotransporter, syndrome from 30 families for CLCNKB mutations, CFTR or the voltage-gated ClϪ channel CLC5 [30–32], showed that mutations in this gene most commonly cause may participate in basolateral ClϪ exit. Of great interest the classic Bartter syndrome [15]. Nevertheless, in a mi- is the recent identification of barttin, a ClϪ channel nority of patients, various mutations in CLCNKB were ␤-subunit that colocalizes with the channel in basolateral found to cause phenotypes that overlapped with either membranes of the renal tubule and inner ear epithelium antenatal Bartter syndrome or Gitelman syndrome. Re- [17, 18]. Barttin appears to mediate basolateral recycling cently, Jeck et al reported three patients whose clinical of ClϪ [18]. course was characterized by gradual transition from CBS The summary of these interrelated transport processes to GS phenotype, and who were found to carry three leads to the hypothesis that variation in expression and/or different mutations in the CLCNKB gene [15]. function of any one of the channels or transporters par- The findings of these studies demonstrating the pheno- ticipating in ClϪ transport in the TAL and the DCT may typic variability associated with CLCNKB mutations could modify to variable degrees the derangement in ClC-Kb be potentially attributed to the nature of the genetic de- function, thereby influencing the disease phenotype. Such fect. For example, differences in the type and/or location a modifying effect could occur at the cellular/regulatory of the mutation within the gene could result in differ- level, whereby one or more transport mechanisms are re- ences in the magnitude of the loss of function of the ClϪ cruited to compensate for impaired ClC-Kb function or, channel, thereby resulting in differences in clinical pic- alternatively, may be determined at the level of modifier tures. Alternatively, age-related alterations in the activ- genes. Our linkage analysis studies have provided no evi- ity of the defective ClϪ channel could lead to a gradual dence that the phenotypic variability in our family was transition from one phenotype to the other, as demon- related to a defect in the Naϩ-Kϩ-2ClϪ cotransporter gene, strated by Jeck et al in their longitudinal observation of SLC12A1 or the thiazide-sensitive NaCl cotransporter three patients [15]. These considerations, however, do not gene, SLC12A3. Obviously, further genetic analysis of apply to the family reported here, in which the phenotypic the family, in an effort to identify such a modifier gene, differences have been observed in a cross-sectional study would be of great importance. Such a study also should in patients bearing the same missense homozygous muta- include a careful analysis of genetic polymorphism in tion presumably originating in a common ancestor. various loci relevant to renal tubular ion transport. How could a specific genetic defect in CLCNKB result Hypomagnesemia and/or hypocalciuria, both typical in such a wide range of clinical phenotypes within the findings in Gitelman syndrome [3, 33], occurred in six same family? Examination of the nature of ClϪ transport (50%) of the affected family members. Of these, four had in the distal parts of the renal tubule potentially could concomitant hypomagnesemia and hypocalciuria. The provide an explanation for this phenomenon. Trans- hypomagnesemia was mild as previously noted in pa- epithelial ClϪ transport in the TAL is a complex process tients with CLCNKB mutations who had this laboratory that involves the coordinated interplay between the lu- abnormality. The exact mechanisms underlying the hy- minal, bumetanide-sensitive Naϩ-Kϩ-2ClϪ cotransporter pocalciuria and hypomagnesemia in Gitelman syndrome (NKCC2), the luminal, adenosine 5Ј-triphosphate (ATP)- are unclear. It has been hypothesized that the loss of regulated, inwardly-rectifying Kϩ channel (ROMK), the function mutation in TSC causes hypocalciuria by the basolateral ClϪ channel (ClC-Kb), as well as other co- same mechanism as thiazide diuretics. According to this transporters and channels [3, 25, 26]. Chloride is re- hypothesis, impaired Naϩ reabsorption across the lumi- absorbed across the luminal membrane of the TAL cell nal membrane of the distal convoluted tubular cell, cou- by the activity of NKCC2. This cotransporter is driven pled with continued exit of intracellular ClϪ through by the low intracellular Naϩ and ClϪ concentrations gen- basolateral ClϪ channels, causes the cell to hyperpolar- erated by the basolateral Naϩ-Kϩ-ATPase and ClC-Kb, ize. This, in turn, stimulates entry of calcium into the respectively. In addition, ROMK enables the functioning cell via luminal voltage-activated Caϩϩ channels [34]. In of NKCC2 by recycling Kϩ back to the renal tubular addition, the lowering of intracellular Naϩ concentration lumen. ClϪ transport in the distal convoluted tubule oc- facilitates Caϩϩ exit via basolateral Naϩ/Caϩϩ exchanger. Zelikovic et al: Mutation in Gitelman and Bartter syndromes 31

The mechanisms whereby a CLCNKB mutation decreases characterized by hypokalemia and hypomagnesaemia. Trans Assoc ϩϩ Am Physicians 79:221–235, 1966 Ca excretion in the urine, as observed in our patients, 5. Simon DB, Karet FE, Hamdan JM, et al: Bartter’s syndrome: Ϫ are unclear. It is possible that impaired Cl exit at the Hypokalemic alkalosis with hypercalciuria, is caused by muta- basolateral membrane of the DCT and subsequent intra- tions in the Na-K-2C1 cotransporter NKCC2. Nat Genet 13:183– Ϫ 188, 1996 cellular accumulation of Cl directly or indirectly influ- 6. Simon DB, Karet FE, Rodriguez-Soriano J, et al: Genetic hetero- ϩϩ ence DCT cell function, thereby increasing Ca reab- geneity of Bartter’s syndrome revealed by mutations in the Kϩ sorption in this tubule segment. The reasons for renal channel, ROMK. Nat Genet 14:152–156, 1996 ϩϩ 7. Simon DB, Bindra RS, Mansfield TA, et al: Mutations in the Mg wasting and hypomagnesemia in Gitelman syn- chloride channel gene, CLCNKB, cause Bartter’s syndrome type drome, in general, and the role of the ClC-Kb defect III. Nat Genet 17:171–178, 1997 in the pathogenesis of hypomagnesemia, in particular, 8. Simon DB, Nelson-Williams C, Bia MJ, et al: Gitelman’s variant of Bartter’s syndrome, inherited hypokalemic alkalosis, is caused remain to be elucidated. Whatever these mechanisms by mutations in the thiazide-sensitive Na-C1 cotransporter. Nat Ϫ might be, it appears that in some patients with a Cl Genet 12:24–30, 1996 channel defect, as observed in our family, specific mecha- 9. Vargas-Poussou R, Feldman D, Vollmer M, et al: Novel molecular variants of the Na-K-2C1 cotransporter gene are responsi- nisms are activated that compensate for the derangement ble for antenatal Bartter syndrome. Am J Hum Genet 62:1332– in mineral transport and prevent the development of 1340, 1998 hypomagnesemia and hypocalciuria. Moreover, some 10. Kurtz CL, Karolyi L, Seyberth HW, et al: A common NKCC2 mutation in Costa Rican Bartter’s syndrome patients: Evidence patients even develop hypercalciuria. The exact nature for a founder effect. J Am Soc Nephrol 8:1706–1711, 1997 of these mechanisms and the level of their operation is 11. International Collaborative Study Group for Bartter-like a subject for further investigation. Syndromes: Mutations in the gene encoding the inwardly rectifying renal potassium channel, ROMK, cause the antenatal variant of In summary, our findings demonstrate intrafamilial het- Bartter syndrome: Evidence for genetic heterogeneity. Hum Mol erogeneity, namely the presence of Gitelman syndrome Genet 6:17–26, 1997 and classic Bartter syndrome phenotypes in a kindred 12. 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