clinical and genetic spectrum of bartter syndrome type 3 · clinical research clinical and genetic...

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Clinical and Genetic Spectrum of Bartter SyndromeType 3

Elsa Seys,* Olga Andrini,†§ Mathilde Keck,†‡ Lamisse Mansour-Hendili,§ǁ

Pierre-Yves Courand,¶**†† Christophe Simian,ǁ Georges Deschenes,‡‡§§ Theresa Kwon,‡‡§§

Aurélia Bertholet-Thomas,ǁǁ Guillaume Bobrie,¶¶ Jean Sébastien Borde,***Guylhène Bourdat-Michel,††† Stéphane Decramer,‡‡‡ Mathilde Cailliez,§§§ Pauline Krug,§§ǁǁǁ

Paul Cozette,¶¶¶ Jean Daniel Delbet,**** Laurence Dubourg,†††† Dominique Chaveau,‡‡‡‡

Marc Fila,§§§§ Noémie Jourde-Chiche,ǁǁǁǁ¶¶¶¶ Bertrand Knebelmann,§§*****Marie-Pierre Lavocat,††††† Sandrine Lemoine,†††† Djamal Djeddi,‡‡‡‡‡ Brigitte Llanas,§§§§§

Ferielle Louillet,ǁǁǁǁǁ Elodie Merieau,¶¶¶¶¶ Maria Mileva,****** Luisa Mota-Vieira,††††††

Christiane Mousson,‡‡‡‡‡‡ François Nobili,§§§§§§ Robert Novo,ǁǁǁǁǁǁ

Gwenaëlle Roussey-Kesler,¶¶¶¶¶¶ Isabelle Vrillon,******* Stephen B. Walsh,†††††††

Jacques Teulon,†‡ Anne Blanchard,§**§§‡‡‡‡‡‡‡ and Rosa Vargas-Poussouǁ§§‡‡‡‡‡‡‡

Due to the number of contributing authors, the affiliations are listed at the end of this article.

ABSTRACTBartter syndrome type 3 is a clinically heterogeneous hereditary salt-losing tubulopathy caused bymutations of the chloride voltage-gated channel Kb gene (CLCNKB), which encodes the ClC-Kb chlo-ride channel involved in NaCl reabsorption in the renal tubule. To study phenotype/genotype corre-lations, we performed genetic analyses by direct sequencing and multiplex ligation-dependent probeamplification and retrospectively analyzed medical charts for 115 patients with CLCNKB mutations.Functional analyses were performed in Xenopus laevis oocytes for eight missense and two nonsensemutations. We detected 60mutations, including 27 previously unreported mutations. Among patients,29.5% had a phenotype of ante/neonatal Bartter syndrome (polyhydramnios or diagnosis in the firstmonth of life), 44.5% had classic Bartter syndrome (diagnosis during childhood, hypercalciuria, and/orpolyuria), and 26.0% had Gitelman-like syndrome (fortuitous discovery of hypokalemia with hypomag-nesemia and/or hypocalciuria in childhood or adulthood). Nine of the ten mutations expressed in vitro

decreased or abolished chloride conductance. Severe (large deletions, frameshift, nonsense, and es-sential splicing) and missense mutations resulting in poor residual conductance were associated withyounger age at diagnosis. Electrolyte supplements and indomethacin were used frequently to inducecatch-up growth, with few adverse effects. After a median follow-up of 8 (range, 1–41) years in 77patients, chronic renal failure was detected in 19 patients (25%): one required hemodialysis and fourunderwent renal transplant. In summary, we report a genotype/phenotype correlation for Bartter syn-drome type 3: complete loss-of-function mutations associated with younger age at diagnosis, and CKDwas observed in all phenotypes.

J Am Soc Nephrol 28: 2540–2552, 2017. doi: https://doi.org/10.1681/ASN.2016101057

Received October 3, 2016. Accepted February 27, 2017.

E.S. and O.A. contributed equally to this work.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Rosa Vargas-Poussou, Hôpital Européen GeorgePompidou, INSERM, UMR970, Paris-Cardiovascular Research Center,20-40 rue Leblanc, 75015 Paris, France. Email: [email protected]

Copyright © 2017 by the American Society of Nephrology

2540 ISSN : 1046-6673/2808-2540 J Am Soc Nephrol 28: 2540–2552, 2017

https://doi.org/10.1681/ASN.2016101057

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mailto:[email protected]

Bartter syndromes (BS) and Gitelman syndrome (GS) are au-tosomal recessive salt-losing tubulopathies caused by defectivesalt reabsorption.They are characterized byhypokalemia,met-abolic alkalosis, and secondary aldosteronism, with normal orlow BP.1,2 BS are classified by phenotype (antenatal or classic)or genotype (types 1–5). Antenatal BS (ABS) is themost severeform, characterized by polyhydramnios, premature birth, life-threatening episodes of neonatal salt and water loss, hyper-calciuria, and early-onset nephrocalcinosis.3 Classic BS (CBS)occurs in infancy or early childhood and is characterized bymarked salt wasting and hypokalemia, leading to polyuria,polydipsia, volume contraction, muscle weakness, growth re-tardation and, sometimes, nephrocalcinosis.4 BS types 1, 2,and 3 are caused by mutations of genes expressed in the thickascending limb (TAL) of the loop of Henle encoding the lu-minal Na+–K+

–2Cl2 cotransporter (SLC12A1; OMIM#601678), the luminal K+ channel ROMK (KCNJ1; OMIM#241200), and the basolateral chloride channel ClC-Kb(CLCNKB; OMIM #607364), respectively.5–7 Loss-of-function mutations of BSND encoding barttin, an essentialb subunit for chloride channels, cause BS type 4a withsensorineural deafness (OMIM #602522).8 Simultaneousmutations of CLCNKB and CLCNKA cause type 4b BS(OMIM #613090).9 Finally, severe gain-of-function muta-tions of the extracellular Ca2+-sensing receptor gene canresult in a Bartter-like syndrome (BS type 5, OMIM#601199).10,11 GS (OMIM #263800) is a milder disease fre-quently associated with hypomagnesemia and hypocalciuria.GS is often asymptomatic or associated with mild symptoms,such as muscle weakness, salt craving, paresthesia, and tetany.GS is related to loss-of-function mutations of the SLC12A3gene encoding the apically expressed thiazide-sensitive NaClcotransporter of the distal convoluted tubule (DCT).12

The first described patients with BS type 3 had a clinicalphenotype corresponding to CBS.7 Considerable phenotypicvariability has since been described: CLCNKB mutations canalso underlie the ABS, neonatal BS (NBS), and Gitelman-like(GLS) phenotypes.13–15 This study aimed to shed light on thephenotypic heterogeneity of BS type 3 by investigating phe-notype/genotype correlations in a very large French cohort,and by evaluation of published results and original data forin vitro expression.

RESULTS

PopulationWe retrospectively analyzed results for 115 patients (56 menand 59 women) from 111 families with CLCNKB mutationsevaluated at the Genetics Department of Georges PompidouEuropean Hospital over the last 15 years. A history of consan-guinity was recorded for 22 families; the geographic origin isshown in Supplemental Tables 1–3.

Initial Clinical PresentationThirty-four patients from 32 families (29.5%) presented withABS/NBS, 51 patients from 49 families (44.5%) presentedwith CBS, and 30 patients from 30 families (26%) presentedwith GLS.

Mutations and Large RearrangementsGenetic status and mutation type were determined for eachinitial phenotype group (Table 1). The detailed genotypes ofeach patient are summarized in Supplemental Tables 1–3. Thedeletion of a single allele was excluded in patients with homo-zygous point mutations and no consanguinity, and molecularabnormalities of the other genes implicated in GS and BS wereexcluded in patients with only one heterozygous mutation.The breakpoints of large rearrangements were not character-ized; in consequence, we cannot exclude the possibility thatpatients with homozygous deletions from nonconsanguine-ous families harbored two different deletions. Testing was car-ried out for both parents in 22 families and only the mother inseven families. In all cases, parents were heterozygous forthe homozygous mutation detected in the proband or forone of the two mutations detected in compound heterozygousprobands.

Sixtydifferentmutationsweredetected: 55%missense, 13%frameshift, 12% nonsense, 10% large deletions, and 10%splice-site mutations (Figure 1). Twenty-seven of these muta-tions were previously unknown (Figure 2, A and B, Supple-mental Tables 1–3). Two of the three splice-site mutationsdisrupt the obligatory consensus donor or acceptor splicesite and were considered pathogenic as likely to causeexon skipping and frameshift. The variant at position 26in the acceptor site of exon 14 is a known rare variant

Table 1. Genetic status of patients according to initial phenotype, and percentages of mutated alleles by phenotype andmutation type

Phenotype

Genetic Status, No. of Patients (%) Type Of Mutation: No. (%) of Mutated Alleles (N=216)

HomozygousCompound

HeterozygousOnly One Heterozygous

MutationLarge

DeletionsSplice-SiteMutations

Frameshift/NonsenseMutation

MissenseMutations

ABS/NBS 22 (65) 10 (29) 2 (6)a 37 (56) 7 (11) 5 (7) 17 (26)CBS 20 (39) 27 (53) 4 (8)b 37 (38) 6 (6) 22 (22) 33 (34)GLS 14 (47) 8 (27) 8 (27)b 12 (23) 0 10 (19) 30 (58)ALL 56 (49) 45 (39) 14 (12) 86 (40) 13 (6) 37 (17) 80 (37)aMutations in SLC12A1, KCNJ1, and BSND were excluded.bMutations in SLC12A3 were excluded.

J Am Soc Nephrol 28: 2540–2552, 2017 Bartter Syndrome Type 3: Clinical and Genetics 2541

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(rs369329893, allele frequency in black populations of 0.02%)for which MaxEntScan predicts a 100% decrease in splice-sitescore and SpliceSiteFinder predicts activation of an introniccryptic acceptor site. Unfortunately, no mRNA from this pa-tient was available for analysis.

Two of the 13 previously unreported missense mutationswere present in the same allele in patient BR050 (p.Arg395Trpand p.Ala469Pro), and p.Gly465Arg was detected in the same

allele as the known mutation p.Pro124Leu in three patients(BR116–1, BR157–1, and GT657–1). Eight of the 13 missensevariations affected conserved amino acids and were predictedby at least four out of five tools used for in silico analysis aspotentially pathogenic. The remaining five missense changes(p.Ser218Asn, p.Ala254Val, p.Arg395Trp, p.Ile447Thr, andp.Ala469Pro) were classed as variations of unknown signifi-cance (VOUS) (Supplemental Table 4). Among these changes,only the p.Arg395Trp has been described in databases(rs34255952) with an allelic frequency of 2% in blacks andhas not been detected in whites. Of the 33 missense mutationsdetected in our population, 13 were previously shown to resultin loss of function.16 In silico predictions are presented in Sup-plemental Table 5 for missense mutations for which in vitroanalysis was not performed.

Functional Expression of ClC-Kb Mutants in XenopusOocytesWe investigated the effect of two new missense mutations pre-dicted tobepathogenic (p.Gly345Ser andp.Ala510Thr), twonew

Figure 1. Type of mutations of CLCNKB detected in patientswith BS type 3 (n=60).

Figure 2. Locations of novel CLCNKB mutations and of mutations expressed in vitro. (A) CLCNKB gene structure, showing the newlydiscovered large deletions and splicing mutations. (B) Schematic topological model of the ClC-Kb protein: the lower part of the modelcorresponds to the intracellular region, and the upper part is extracellular. Each rectangle represents one of the 18 a-helices and thetwo cystathionine-b-synthase (CBS) domains. The a-helices involved in the selectivity filter, those interacting with Barttin, and thoselocated at the dimer interface are shown in blue, green, and pink, respectively. Previously unknown missense ( ) and nonsense ( )mutations are shown in red; previously described mutations are shown in blue ( ); mutations expressed in vitro are underlined.

2542 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2540–2552, 2017





VOUS (p.Arg395Trp and p.Ala469Pro), four previouslydescribed missense mutations (p.Gly296Asp, p.Ser297Arg,p.Gly424Arg, and p.Gly433Glu) and two nonsense mutations(p.Trp391Ter and p.Arg595Ter) on chloride conductance inXenopus oocytes; p.Gly424Arg and p.Gly433Glu are locatedina-helix N of ClC-Kb, which is involved in the selectivity filter;p.Gly296Asp and p.Ser297Arg are located in thea-helix J, whichinteracts with barttin; p.Ala510Thr is found in the a-helix Q,involved in the dimer interface; and Arg595Ter is present inthe CBS1 domain involved in channel common gating and traf-ficking. The p.Gly345Ser and p.Ala469Pro mutations affecta-helices K and O, respectively, and the p.Trp391Ter andp.Arg395Trp mutants affect the L-M linker (Figure 2B). Nineof these ten mutations significantly decreased or abolished nor-malized conductance (Figure 3). The p.Trp391Ter, p.Gly296Asp,p.Gly424Arg, p.Gly433Glu, p.Ala469Pro, and p.Arg595Ter muta-tions abolished conductance, whereas p.Ser297Arg, p.Gly345Ser,and p.Arg395Trp decreased conductance to 61%, 57%, and 65%of wild-type values, respectively (significantly different fromoocytes expressing wild-type ClC-Kb and noninjected oocytes).

By contrast, p.Ala510Thr had no influence on channel conduc-tance. Finally, the p.Arg395Trp/p.Ala469Pro double mutation de-creased conductance to 37% of wild-type values.

Clinical Data at DiagnosisTable 2 summarizes clinical and biochemical characteristics atbirth and at diagnosis. As expected, gestational age (GA) atbirth was significantly lower in the ABS/NBS group than in theCBS and GLS groups, but similar between the CBS and GLSgroups. Age at diagnosis was significantly lower in the ABS/NBS group than in the other two groups and in the CBS groupthan in the GLS group. Polyhydramnios was found in 29 pa-tients with ABS/NBS (85%), at mean GA of 28 weeks, andamniotic fluid had to be drained in four patients. Ten patients(five ABS/NBS and five CBS) had birth weights below the 10thpercentile, and four patients (two ABS/NBS and two CBS) hadbirth heights below the 10th percentile for GA at birth.

Plasma sodium and chloride concentrations were signifi-cantly lower and plasma renin andmagnesium concentrationswere significantly higher in CBS and ABS/NBS groups than in

the GLS group; plasma potassium and totalCO2 concentrations were similar in allgroups. Strong hypochloremia is a knownphenotypic hallmark of BS type 3.17–19 Wetherefore compared the relationship be-tween plasma sodium and chloride con-centrations between patients with BS types1 and 2 (n=21) and patients with BS type 3(n=51). This curve was shifted downwardin the BS type 3 group, indicating thatplasma chloride depletion could not be ac-counted for by hyponatremia (Figure 4).No difference was observed in parametersat diagnosis when confirmed homozygouspatients are compared with compoundheterozygous patients (data not shown).

Clinical and Biologic Data duringFollow-UpClinical manifestations during follow-upand treatmentwere recorded for 77 patients(Table 3). Median follow-up was 8 yearsand was similar in the three groups. Themain treatments administered to these pa-tients were NaCl and KCl supplementationand nonsteroidal anti-inflammatory drugs(mainly indomethacin). The main adverseeffects were abdominal pain (n=5), weightgain (n=1), esophagitis (n=1), and diar-rhea (n=1). One of the main criteria of asuccessful treatment is a normal growth; inthis cohort, 63 out of 77 patients (82%)had a height between 22 SD and +1 SDor a normal height as adults. Fourteen pa-tients had height below 22 SD: five

Figure 3. Functional studies of selected ClC-Kbmutants (n=10). Conductance at +60mVfor noninjected oocytes (NI) and for oocytes into which mutant ClC-Kb cRNA wasinjected, normalized with respect to the mean value for wild-type (WT) ClC-Kb andexpressed as the mean6SEM. The mutants were exposed to a solution at pH 7.4containing 10 mM Ca2+ (A) or to a solution at pH 9.0 containing 20 mM Ca2+ (B). AsClC-Kb current increases at high external Ca2+ concentration or high pH, these so-lutions were chosen to obtain a submaximal current. Number of measurements for(A): NI (n=63), WT (n=109), p.Trp391Ter (n=12), p.Arg395Trp (n=16), p.Arg395Trp/p.Ala469Pro (n=20), p.Gly424Arg (n=16), and p.Ala469Pro (n=10). Number of mea-surements for (B): NI (n=9), WT (n=16), p.Gly122Val (n=6), p.Gly296Asp (n=5),p.Ser297arg (n=7), p.Gly345Ser (n=8), p.Gly433Glu (n=8), p.Ala510Thr (n=8), andp.Arg595Ter (n=3). $P,0.05 for the difference between NI or mutant ClC-Kb andWT; *P,0.05 for the difference between WT or mutant ClC-Kb and NI.



Table

2.Clin

ical

andbiologic

man

ifestations

atdiagno

sis

Variable

ABS/NBS(n=34

)CBS(n=51

)GLS

(n=30

)

PValue

All

ABS/N

BS

versus

CBS

ABS/NBS

versus

GLS

CBS

versus

GLS

Ageat

diagno

sis,yr

0.1(0.02–

0.66

)1(0.4–9.6)

528

(18–

42)2

,0.00

1,0.00

1,0.00

1,0.00

1GAat

birth,

wk

37(36–

40)6

40(39–

40)16

40(40–

40)12

0.00

10.00

20.00

1ns

GAat

onseto

fpolyh

ydramnios,w

k28

.25(22–

33)22

Weight

atbirth,

g27

90(247

0–33

80)8

3200

(285

0–35

00)23

3100

(296

0–33

35)27

nd—

——

Plasmaco

ncen

trations

Sodium,1

33–14

6mmol/L

134(131

–13

6)12

133(127

–13

7)24

138(136

–14

0)17

nd—

——

Potassium,3

.5–5mmol/L

2.7(2.4–3.1)

52.6(2.3–2.9)

15

2.8(2.5–3.0)5

0.21

——

—

Chlorid

e,90

–11

7mmol/L

87(82–

95)11

87(74–

95)24

97(94–

99)12

0.00

2ns

0.00

10.00

3CO

2t,18

–25

mmol/L

33(28–

37)9

32(28–

36)18

31(30–

33)12

0.53

——

—

Calcium

,2.2–2.6mmol/L

2.67

(2.49–

2.80

)12

2.54

(2.38–

2.64

)25

2.39

(2.32–

2.52

)13

0.00

1ns

0.00

10.01

Mag

nesium

,0.75–

1mmol/L

0.95

(0.87–

1.00

)17

0.91

(0.76–

1.00

)22

0.77

(0.69–

0.81

)14

0.00

1ns

0.00

1ns

Renin,

times

theno

rmal

upper

limitfora

ge

11(6–26

)16

3(1–15

)26

1(0–3)

14

0.00

1ns

,0.00

10.02

Aldosteron

e,tim

estheno

rmal

upper

limit

0.4(0.3–2.0)

15

2.0(0.8–2.5)

30

0.6(0.2–1.3)

16

nd—

——

Urin

arycalcium-to-crea

tinineratio

,Zvalue

1.23

(0.27–

2.13

)13

0.70

(0.11–

1.45

)31

20.37

(22.77

–0.62

)21

ndNep

hrocalcinosis,%

29.4

143

0.01

0.1

0.01

0.25

Mainsymptoms,%

Failu

re-to-thriv

e47

2413

0.01

0.04

0.00

40.19

Polyuria

5642

230.03

0.25

0.01

0.07

Asthe

nia/cram

ps

02

200.02

0.41

0.04

0.06

Fortuitous

disco

very

ofhy

pok

alem

ia0

040

,0.00

11

,0.00

1,0.00

1

Value

sareex

pressed

asmed

ians

andinterqua

rtile

intervals.Su

perscrip

tvalue

sco

rrespon

dto

thenu

mber

ofmissing

data.Calcium

-to-crea

tinineratio

isex

pressed

asaZvaluerelativ

eto

theno

rmalvalues

fora

ge;

hyperca

lciuria

isdefi

nedbyaZva

lue.2.

nd,n

otdon

e,.50

%of

thedatamissing

inon

eor

moregroup

s;—

,notd

one;

CO

2t,totalcarbon

dioxide.



patients with ABS/NBS, including one with a growth hormone(GH) deficiency (IGF1=38 ng/ml before GH initiation at 15 yearsof age); eight patients with CBS, including two with GH de-ficiency and two with CKD; and one patient with GLSwith noidentified cause of failure-to-thrive.

Abnormalities in psychomotor and neurologic develop-ment included psychomotor retardation in four patientswith ABS/NBS and four patients with CBS (Table 3). Fivepatients with CBS required psychiatric follow-up (hyperactiv-ity, anorexia, or eating disorders).

Irregular heart rate orECGabnormalitiesweredocumentedin six patients: one patient with ABS/NBS had premature ven-tricular beats with prolonged QT interval, three patients withCBS had a right bundle branch block or U wave, and onepatients with GLS presented with torsade de pointe attacks.None of the patients in this cohort had high BP.

Fourteen patients developed nephrolithiasis or nephrocal-cinosis during follow-up. None of the patients required shock-wave lithotripsy. Other renal and urological abnormalitiesdiagnosed in eight patients with ABS/NBS and nine patientswith CBS are detailed in Table 3. Proteinuria data wereavailable for 43 patients, nine of whom displayed glomerularproteinuria .50 mg/dl.

Nineteen (tenwomenandninemen)of the 77 patients (25%)presented with CKD (Table 4). Ten patients presented with stage2CKD: four patientswithABS/NBS, four patientswithCBS, andtwo patients with GLS. Two patients reached stage 3 CKD (onepatient with ABS/NBS and one patient with GLS). One patientwith CBS reached stage 4 CKD. Six patients reached stage 5 CKD(three patients with ABS/NBS and three patients with CBS), at amean age of 25 (6–49) years. Renal biopsies were performed infive out of 19 patients with CKD. Four patients with stage 5 CKD

Figure 4. Correlation between plasma sodium and plasma chlorideconcentrations in patients with BS type 1 and 2 (black symbols, grayarea) and in patients with BS type 3 (white symbols, orange area).

Table 3. Associated clinical manifestations and treatment during follow-up, for 77 patients with CLCNKB mutations

Variable ABS/NBS (n=26) CBS (n=35) GLS (n=16)

Follow-up duration, median (range) 7.5 (1–36) 8 (1–41) 8 (1–28.5)Growth retardation (below 22 SD) 5 8 1NeurologicPsychomotor retardation 4 4 —

Psychiatric follow-up — 5 —

Speech therapy 3 4 —

Neurologic follow-up 2 2 1Renal abnormalitiesRenal hypoplasia 2 — —

Hyperechogenic kidney 2 4 —

Cyst 2 3 —

Urological abnormalitiesHydroureteronephrosis 1 1 —

Vesico-ureteral reflux — 1 —

Anterior urethral valves 1 — —

Proteinuria$50 mg/dl 4/17 4/17 1/8Nephrolithiasis 2 5 —

Nephrocalcinosisa 3 4 —

Cardiovascular manifestations irregular heart rate orECG abnormalities (details in the text)

1 3 1

TreatmentContinuous enteral nutrition 7 4 —

NaCl supplementation 21 17 —

KCl supplementation 24 27 6—, None; ECG, Electrocardiogram; NaCl, Sodium Chloride; KCl, Potassium Chloride.aDetected during follow-up and reported in the medical chart.



Table

4.Clin

ical

characteristicsof

patientswithBStype3an

dCKD

Phe

notype

Mutations

Class

Sex

Weight

atBirth,k

gAgeat

Diagno

sis

Ageat

Last

Control

eGFR

,aml/min

per

1.73

m2

PU

NC(yr)

Other

(yr)

Indomethac

in(yr)

Ren

alBiopsy

(age,

yr)

CKD

stag

e2

ABS/NBS

CL/PL

F3.13

2yr

7.5

76ND

Yes(2)

Hyd

roureterone

phros

is,A

PYe

s(1.5)

No

ABS/NBS

CL/CL

F2.86

117

85Ye

s,,2g/L

No

Cortic

alcyst

Yes(16)

Yes(2)

ABS/NBS

CL/CL

F1.90

2mo

1165

No

No

No

Yes(12)

No

ABS/NBS

CL/CL

MND

1mo

4269

ND

No

Yes(N

D)

No

CBS

CL/CL

FND

7yr

1680

ND

bHyp

ercalciuria

,bon

edem

ineralization

No

No

CBS

PL/C

LM

ND

2mo

3381

Yes(26)

Yes(30)

No

CBS

CL/CL

M3.2

5mo

682

No

No

No

Yes(3)

No

GLS

PL/PL

FND

43yr

5676

No

No

No

No

GLS

?/?

MND

46yr

4879

No

ND

No

Yes(5)

No

GLS

CL/CL

FND

30yr

5075

No

No

Psychiatric

disorder

No

No

CKD

stag

e3

ABS/NBS

CL/CL

M3.45

0.01

919

45Ye

s,.2g/L

No

Osteo

pen

iaYe

s(4)

No

GLS

CL/CL

FND

3039

31No

No

Rena

lcysts

No

CKD

stag

e4

CBS

CL/CL

M3.2

0.16

2027

ND

No

Yes(15)

No

CKD

stag

e5

ABS/NBS

CL/CL

M2.76

0.03

6.5

12Ye

s,,2g/L

No

Grisce

llisynd

rome+rena

lhy

pop

lasia

tran

splantation(6.5)

Yes(5)

Bila

teral

nephrec

tomy

ABS/NBS

?/?

F3.45

0.25

26ES

RD

at23

.4Ye

s,.2g/L

No

Hyp

eruricem

iatran

splantation(26)

Yes(5.5)

Yes(23)

ABS/NBS

?/?

MND

0.33

24ES

RD

at24

Yes,,2g/L

Yes

Materna

lalcoho

labuse,

neon

atal

seizures

Yes(10)

Yes(28)

CBS

PL/PL

FND

0.5

16ES

RD

at12

ND

Yes

Tran

splantation(13)

Yes(N

D)

No

CBS

PL/PL

MND

849

13Ye

s,.2g/L

Yes(41)

Nep

hrolith

iasis

Yes(2)

Yes(41)

CBS

CL/CL

F3.5

0.16

29ES

RD

at28

Yes,.2g/L

bTran

splantation(29)

Yes(N

D)

Yes(27.3)

CL:largedeletions,trunc

ating(unlesscarbox

y-term

inal),essentialsplicing,m

issenseno

ncond

uctin

g,and

mutan

tswith

residua

lcurrents,20

%.P

L:missensemutan

tswith

residua

lcurrents.21

%.D

etailedresults

ofrena

lbiopsies

aredescribed

inSu

pplemen

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had diffuse glomerular and tubulointerstitial lesions with en-larged glomeruli presenting with FSGS. One patient with stage2 CKD had minimal glomerular and tubular alterations (Sup-plemental Table 6, Table 4). Patients with CKD were older thanpatients without CKD; they did not differ in terms of birthweight, AINS treatment, urologic or renal abnormalities, orhypokalemia severity (Table 5).

The last eGFR follow-up data for 30 patients with BS type 1,34 patients with BS type 2, and 11 patients with BS type 4awerecompared with eGFR for the 77 patients with BS type 3. Thesegroups had similar age distribution, and eGFR decreased withage (Supplemental Figure 1). In patients with BS type 1 and BStype 4, eGFR decrease was more severe and there were higherproportions of patients with CKD 3–5 than in patients with BStype 2 and BS type 3 (Supplemental Figures 1 and 2).

Genotype/Phenotype CorrelationLarge deletions were more frequent in patients with earlieronset, and severe phenotypes and missense mutations weremore common in the GLS phenotype (Table 1). Similar resultswere obtained if other potentially severe mutations (frame-shift, nonsense, and essential splicing) were considered withlarge deletions: severe mutated alleles were more frequent inpatients with ABS/NBS and CBS (74 and 66% respectively)than in patients with GLS (42%). Further, missense mutationswere more frequent in patients with less severe phenotypes:58% in patients withGLS versus 34% and 26% in patients withCBS and ABS/NBS, respectively.

We classified mutations into two groups: complete loss-of-function (CL) andpartial loss-of-function (PL) groups (Table 6).We included p.Trp610Ter, the only C-terminus–truncatingmutation expressed in vitro and yielding a residual current.20

Each mutated allele was classified separately, independently ofthe initial phenotype, and only patients for whom both allelescould be classified were analyzed (n=85) (Supplemental Tables1–3). ClC-Kb functions as a homodimer. The residual activityof the CL/PL genotypes may therefore correspond to homo-dimers of PLmutants, with similar consequences to the PL/PLgenotype.We therefore analyzed the CL/PL genotype togetherwith the PL/PL genotype. With this classification, 56 patientshad a CL/CL genotype, and 29 patients had a CL/PL or PL/PLgenotype. CL/CL genotypes were associated with a signifi-cantly younger age at diagnosis than CL/PL and PL/PL geno-types. No difference was observed for the other biologicparameters analyzed (Table 6).

DISCUSSION

BS are phenotypically and genotypically heterogeneous. Phe-notype/genotype correlations highlighting the link betweenparticular traits and genetic types (i.e., transitory hyperkale-mia in BS type 2, severe hypochloremia in BS type 3, andhearing loss in BS type 4) have been identified in previousstudies.17–19 BS type 3 is particularly heterogeneous in terms

of clinical presentation, accounting for the diverse initial di-agnoses attributed (ABS/NBS, CBS, or GLS). We investigatedthe basis of this variability in a cohort of 115 patients harbor-ing CLCNKBmutations, and studied the phenotype/genotypecorrelation on the basis of clinical presentation and follow-upas well as on in vitro functional studies of missense mutants.

More than 54 mutations of this gene have been reported infree access HGMD (www.hgmd.cf.ac.uk) and scientific publi-cations.4,7,17,19,21–25 They include a high frequency of largerearrangements favored by the close location of the homolo-gous CLCNKA. We detected 60 different mutations, 27 ofwhich had not been previously reported (13 missense, fiveframeshift, three nonsense, three splice-site mutations, andthree large deletions). Thirteen of thesemutations (frameshift,nonsense, splice-site mutations, and large deletions) were pre-dicted to result in the production of unstable mRNAs ortruncated or absent proteins. Eight of the 13 previouslyunknownmissensemutations were predicted to be pathogenicin silico (Supplemental Table 4). Three out of the other five,classified as VOUS, were expressed in Xenopus laevis oocytes(p.Arg395Trp, p.Ala469Pro, and p.Gly345Ser) as were two pre-viously described mutations (p.Gly424Arg and p.Gly433Glu)detected as the only heterozygous mutation in two patients.All of these mutations significantly decreased chloride conduc-tance. The p.Ala510Thr, predicted in silico as pathogenic, had achloride conductance similar to that of the wild-type channel.The molecular abnormality of patient heterozygous for thisvariant thus remains unidentified.

In this large BS type 3 cohort, we confirmed the phenotypicvariability, consisting of about 30% ABS/NBS, 45% CBS, and25% GLS (Table 2). In order to determine if the type of mu-tation influences the phenotype, we first correlated them withinitial clinical presentation. Large deletions and severe muta-tions were associated with all clinical presentations, but weremore frequent in ABS/NBS and CBS. Next, 85 patients withtwo mutated alleles were analyzed for phenotype/genotypecorrelations, taking into account the type of mutation and invitro expression results, regardless of initial clinical presenta-tion. Patients with complete loss-of-function (CL/CL) weresignificantly younger at diagnosis than patients harboringone or two alleles with a partial loss-of-function (CL/PL orPL/PL), suggesting that the type ofmutationmay influence theclinical presentation of BS type 3.

Surprisingly, the milder GLS phenotype did occur in pa-tients harboring severe mutations or deletions, suggesting thatthe phenotype severity is not only driven by CLCNKB allelicvariability. Phenotypic heterogeneity of BS type 3 has beenattributed to distribution of the ClC-Kb channel along thenephron and to possible compensatory function of the ClC-Ka channel. ClC-Kb channel is expressed in the TAL, DCT,and collecting duct, where it transfers chloride (Cl2) ions tothe basolateral side.26 Impaired ClC-Kb function in the TALresults in lower levels of Cl2 exit, NaCl reabsorption throughthe Na-K-2Cl cotransporter, and divalent cation reabsorption,accounting for the Bartter phenotype. Defective basolateral









http://www.hgmd.cf.ac.uk


Cl2 exit in the DCT decreases NaCl reabsorption via the NaClcotransporter, accounting for the GLS phenotype in other pa-tients. Two recent studies in which the mouse Clcnk2 gene (cor-responding to CLCNKB in humans) was disrupted confirmedthat ClC-K2 is the principal chloride channel in all three neph-ron segments and that TAL impairment is not compensated byClC-K1 (corresponding to the human ClC-Ka channel, which isalso expressed in the TAL).27,28 Nevertheless, it cannot be ex-cluded that allelic variants of genes encoding KCl cotransportersor other chloride channels may also compensate for renal so-dium loss, accounting for phenotypic variability.27,29

Next generation sequencing (NGS) approaches allow par-allel analysis of several genes, which is particularly useful indiseases with genetic heterogeneity, such as ABS, or in diseaseswith phenotypic variability such as BS type 3. A genetic con-firmation is important for the follow-up as well as to improve

our knowledge of the natural history of these syndromes.Therefore, we recommend the use of NGS panels to diagnosisconfirmation. NGS could also be useful to determine whetheradditional genes are involved in the observed clinical variabil-ity. In the future, in vitro studies of additional mutant proteinscan contribute to improving our understanding of the pheno-type/genotype correlation and of the precise pathogenicmechanism of mutants. These studies have the potential in-terest to define targeted therapeutic approaches, such as chan-nel openers or pharmacologic chaperones.30,31

Despite missing data for some phenotypic criteria becauseof the retrospective nature of this study, several patternsemerged from our analysis. First, growth retardationwas com-mon but frequently improved with treatment. Fourteen pa-tients presented with persistent growth retardation; two ofthesepatients hadCKD, a commoncause of growth retardation

due to a combination of abnormalities ofthe growth hormone axis, vitamin D defi-ciency, hyperparathyroidism, inadequatenutrition, and drug toxicity; and three pa-tients presented with GH deficiency.32 BSand potassium deficiency have alreadybeen reported to be associated with GH de-ficiency.33–35 One previous study showedthat GH and IGF1 did not stimulate longi-tudinal growth unless hypokalemia wascorrected.36 In two patients with hypoka-lemia (median, 2.5 mmol/L), growthimproved after GH supplementation butremained below 22 SD.

Second, hypochloremia is a hallmark ofBS type 3: an analysis of the data available atdiagnosis showed that hypochloremia wasmore severe in patients with ABS/NBS andCBS than in patients with GLS. ClC-Kb isexpressed not only in the diluting segmentbut also in the intercalated cells of the col-lecting duct. Defects in this segment mayimpair chloride exit and transepithelialchloride reabsorption through the pendrin

Table 5. Patients with BS type 3: Comparison between patients with CKD and patients with normal GFR

Variable CKD Stages 2–5, n=19 eGFR>90 ml/min per 1.73 m2, n=58 P Value

Birth weight, g 3200 (2810–3450)10 2995 (2530–3380)24 0.50Age at diagnosis, yr 0.41 (0.16–8.00) 1.45 (0.39–15.75) 0.18Age at last follow-up, yr 20 (12–42) 13 (6–26) 0.02NSAIDs, n (%) Yes 14 (79) 29 (50) 0.10

No 5 (21) 29 (59)Treatment duration, yr 5.2 (3.2–14.25)2 6 (3.3–9.2)3 0.53Other renal and urologicalabnormalities (excluding nephrocalcinosis)

Yes 5 4 0.24No 12 27

Hypokalemia ,3 with treatment Yes 8 20 1No 8 21

Quantitative values are expressed as medians and interquartile intervals. Superscript values correspond to the number of missing data. NSAIDs, nonsteroidal anti-inflammatory drugs.

Table 6. Characteristics of patients according to genotype severity

VariablePL/CL or PL/PL CL/CL P Value

n=29 n=56

Clinical presentation, % ,0.001ABS/NBS 13.8 41.1CBS 51.7 42.9GLS 34.5 16.1

Age at diagnosis, yr 7.5 (0.7–36)1 0.6 (0.1–6.2)4 ,0.001GA at birth, wk 40 (39.8–40)12 40 (37–40)11 0.42Plasma, n valuesSodium, 133–146 mmol/L 136 (129–140)15 134 (130–137)24 ndPotassium, 3.5–5 mmol/L 2.8 (2.3–3.0)8 2.7 (2.3–3.0)8 0.94Chloride, 90–117 mmol/L 94 (83–97)16 89 (75–95)20 ndCO2t, 18–25 mmol/L 33 (30–35)12 32 (28–37)15 0.73Calcemia, 2.2–2.6 mmol/L 2.5 (2.4–2.8)16 2.6 (2.4–2.7)21 ndMagnesemia, 0.75–1 mmol/L 0.84 (0.77–1.00)11 0.91 (0.82–1.00)28 0.4Renina 3 (2–24)15 8 (2–19)24 0.43

CL: large deletions, truncating (unless carboxy-terminal), essential splicing, missense nonconducting,and mutants with residual currents ,20%. PL: missense mutants with residual currents .21%. Valuesare expressed as medians and interquartile intervals. Superscript values correspond to the number ofmissing data. The P values for continuous variables are for comparisons of the two groups with theMann–WhitneyU test. The P values for dichotomous variables were determined in chi-squared tests orFisher test as appropriate. nd, not done (.50% of the data missing in one or more groups), CO2t, totalcarbon dioxide.aTimes the normal upper limit for age.



Cl/HCO3 exchanger, potentially accounting for the strongerchloride depletion in patients with BS type 3 than in patientswith BS type 1 or BS type 2.19 We found a downward shift ofthe relationship between plasma chloride and sodium concen-trations consistent with a defect in adaptation to chloride de-pletion in patients with BS type 3 as compared with patientswith BS type 1 and BS type 2 patients. These results are con-sistent with the phenotype of mice with Clcnk2 disruption,27

and suggest that sodium and potassium supplementationshould be provided as chloride salts in patients with BS type 3.

Third, 19 patients presented with CKD, and seven of thesepatients also had proteinuria (Table 4). Five patients under-went renal biopsy, which revealed diffuse glomerular andtubulointerstitial lesions with enlarged glomeruli in fourpatients, suggesting compensatory hypertrophy to nephronreduction (Supplemental Table 6, Table 4). Six patients diag-nosed before the age of 8 years displayed progression to ESRDat a median age of 24 years, associated with FSGS in fourpatients. Proteinuria, a lowGFR, and FSGS have been reportedin patients with BS and GS.22,37–39 It has been suggested thatFSGS is a secondary lesion because of adaptation to salt loss,resulting in chronic stimulation of the renin-angiotensin sys-tem.37,39,40 In this study, FSGS occurred in late-stage CKD,suggesting a large contribution of nephron reduction. Wefailed to identify other risk factors of CKD progression, in-cluding birth weight, age at diagnosis, long-term nonsteroidalanti-inflammatory drug treatment, persistent hypokalemia,and other renal abnormalities (Table 5). CKD has been de-scribed in other types of BS.19,22 In our BS cohort, CKD wasobserved in all BS types but the proportion of patients withpreserved renal function (i.e., eGFR.90 ml/min per 1.73 m2)was higher in patients with BS types 2 and 3 and the propor-tion of patients with moderate to severe kidney disease (i.e.,eGFR,60ml/min per 1.73m2) in patients with BS types 1 and4, suggesting that the later BS subtypes 1 and 4 are associatedwith more severe renal prognosis. The mechanism of CKDdevelopment is probably multifactorial, and its elucidationwill require prospective studies. Case reports are rare for pa-tients with BS undergoing renal transplantation. The post-transplantation period was uneventful in our four patients,with the complete disappearance of BS and no recurrence ofFSGS, as previously described.41–43

In conclusion, BS type 3 syndrome, which is caused byCLCNKB mutations, is highly variable phenotypically. Weshow, for the first time, that there is a correlation betweensevere mutations and a significantly younger age at diagnosis,suggesting that milder defects of ClC-Kb function may ac-count for some of this variability. We also confirm the severechloride depletion previously observed in patients with BStype 3 and report that 25% of patients suffer from CKD.Long-term prospective follow-up of this cohort will identifyother severity parameters involved in this genotype/pheno-type correlation, and will allow us to evaluate whether earlydiagnosis and treatment have an influence on the evolutionto CKD.

CONCISE METHODS

PatientsThe study included 115 patients (from 111 families) with CLCNKB

mutations referred to the Genetics Department of Georges Pompidou

European Hospital (Paris, France) from January of 2001 to December

of 2014 for genetic analysis after the diagnosis of BS or GS. The study

was approved by the Comité de Protection des Personnes, Paris-Île de

France XI (reference no. 09069) and informed consent for genetic

studies was obtained from each proband or from their parents (for

minors). Genetic investigations were performed after the clinical and

biologic diagnosis of salt-losing tubulopathy. Patients with a history

of polyhydramnios or clinical manifestations in the first month of life

were considered to have ABS/NBS. Patients diagnosed during child-

hood, with hypercalciuria and/or polyuria, were considered to have

CBS, and children, adolescents, or adults for whom hypokalemia and

hypomagnesemia and/or hypocalciuria were discovered fortuitously

were considered to have GLS. Genetic investigations were extended to

both parents in 22 families and to the mother only in another seven

families. Twenty-three patients from this cohort have been described

before19,23,25 (Supplemental Tables 1–3).

Detection of Point MutationsDNAwas extracted with a salt-basedmethod or with bloodDNAmidi

kits (Qiagen, Venlo, The Netherlands). CLCNKB exons and flanking

intron sequences were amplified by PCR, sequenced with BigDye

Terminator v3.1 cycle sequencing kits, and run on an ABI Prism

3730XL DNA Analyzer (Perkin Elmer Applied Biosystems, Foster

City, CA), as previously described.19,23 DNA mutations were identi-

fied with Sequencher software, by comparison with the reference se-

quence for CLCNKB: NM_000085.4. Each mutation was confirmed

by sequencing a second independent PCR product.

Detection of Large RearrangementsLarge rearrangements were detected by quantitative multiplex PCR

of short fluorescent fragments before June of 2010, and by multiplex

ligation-dependent probe amplification (MLPA) thereafter. We adapted

the quantitative multiplex PCR of short fluorescent fragments method

for the detection of large deletions of CLCNKB.44 The procedure is

described in detail in the Supplemental Material and the primers

used, covering all exons, are listed in Supplemental Table 7. For MLPA,

we used the SALSA MLPA P266-B1 CLCNKB Kit (MRC Holland,

Amsterdam, The Netherlands). The P136 Kit contains 29 probes:

probes for 14 of the 20 exons of CLCNKB (exons 4, 7, 9, 12, 16, and

20 are not represented), four probes for upstream genes (PRM2,

CASP9, and the homologousCLCNKA gene), and 11 reference probes.

The procedure is described in detail in the Supplemental Material.

Bioinformatic Analysis of MutationsThe software used to interpret variants is described in the Supple-

mental Material.

Functional Expression in X. laevisVoltage clamp experimentswere performed as previously described in

X. laevis oocytes.23,25 We injected 10 ng ClC-Kb cRNA and 5 ng










barttin cRNA into defolliculated oocytes, which were then incubated

in modified Barth solution at 16°C. Two-electrode voltage clamp

experiments were performed at room temperature with TURBO

TEC-10CX (npi electronic GmbH, Tamm, Germany) and PClamp

8 software (Axon Instruments, Union City, CA), 2 to 3 days after

injection. Conductance at +60 mV (G+60 mV) was calculated by di-

viding the current at +60 mV by the difference in current between

+60 mV and the reversal potential.

Statistical AnalysesClinical data were analyzed with GraphPad Prism Software (La

Jolla, CA) and SPSS software, release 20.0.0 (SPSS, Chicago, IL).

Kruskal–Wallis tests were used to compare the three groups

(ABS/NBS, CBS, and GLS). In cases of statistical significance,

Mann–Whitney U tests were used to compare the groups in pairs.

Dichotomous variables were compared using the chi-squared test

or Fisher exact test as appropriate. Variables for which .50% of

the data were missing in one group were excluded from the anal-

ysis. Clinical manifestations were analyzed by descriptive meth-

ods, using the number of subjects for each group. Data for in vitro

X. laevis studies were analyzed by ANOVA and Holm–Sidak tests,

with Sigma Stat software.

ACKNOWLEDGMENTS

We thank the patients and their families for agreeing to participate

in this study. We thank all of the staff of the genetics laboratory

ofGeorgesPompidouEuropeanHospital and all of thedoctors involved:

Hans-Jacob Bangstaf (Norway), Pierre Bataille (Boulogne-sur-Mer,

France), Mohammed-Najib Bensemlali (Morocco), Delphine Brouet

(Lorient, France), Christophe Bouaka (Martigues, France), Marina

Charbit (Paris, France),GabrielChoukroun(Amiens, France),Renaud

De La Faille (Bordeaux, France), Geneviève Dumont (Orleans,

France), Olivier Dunand (La Reunion, France), Catherine Dupré-

Goudable (Toulouse, France), Claire Garandeau (Nantes, France),

Angela Granadas (Madrid, Spain), Steven Grange (Rouen, France),

Jean-Christophe Hebert (Guadeloupe, France), Aurélie Hummel

(Paris, France), Khalid Ismaïli (Brussels), Cynthia Kahil (Thonon les

Bains, France), Christina Kanaka (Athens, Greece), Bruno Legallicier

(Rouen, France), Renaud Leray (Montpellier, France), Bruno

Maranda (Sainte Foy, Quebec, Canada), Mohammed-Diab Mahmoud

(La Roche Sur Yon, France), Pierre Merville (Bordeaux, France), Bruno

Moulin (Strasbourg, France), Isabelle Raingeard (Montpellier,

France), Mehta Sanjay (Toronto, Canada), David Schwarz (Zurich,

Switzerland), Pascale Siohan (Quimper, France), Ivan Tack (Toulouse,

France), Cécile Vigneau (Rennes, France), and João Esteves (Ponta

Delgada, Açores, Portugal).

J.T.’s group is funded by a grant from l’Agence Nationale de la

Recherche (grant no. ANRBLANC14-CE12-0013-01/HYPERSCREEN).

This work was supported by the FrenchMinistry of Health (PlanMaladies

Rares) and the European Community (grant nos. FP7EUNEFRON

201590 and EURenOmics 2012-305608).

DISCLOSURESNone.

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This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2016101057/-/DCSupplemental.





AFFILIATIONS

*Pediatric Nephrology Unit, American Memorial Hospital, Reims University Hospital, Reims, France; †Unité Mixte de Recherche en Santé1138, Team 3, Université Pierre et Marie Curie, Paris, France; ‡Institut National de la Santé et la Recherche Médicale, Unité Mixte deRecherche en Santé 872, Paris, France; §Faculté de Médecine, Université Paris Descartes, Paris, France; ǁDepartment of Genetics and¶Centre d’Investigation Clinique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France;**Cardiology Department, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France; ††Centre de Recherche en Acquisition etTraitement de l’Image pour la Santé; Centre National de la Recherche Scientifique Unité Mixte de Recherche 5220; Institut National de laSanté et la Recherche Médicale, Unité 1044; Institut National de Sciences Appliquées-Lyon; Université Claude Bernard Lyon 1, France;‡‡Pediatric Nephrology Unit, Hôpital Robert Debré, Assistance Publique-Hôpitaux de Paris, Paris, France; §§Centre de Référence desMaladies Rénales Héréditaires de l’Enfant et de l’Adulte, Paris, France; ǁǁNéphrogones, Centre de Référence des Maladies Rénales Rares,Pediatric Nephrology, Rhumatology and Dermatology Unit, Hôpital Femme-Mère-Enfant and ††††Exploration Fonctionnelle Rénale etMétabolique, Groupement Hospitalier est Hôpital Edouard Herriot, Hospices Civils de Lyon, Lyon, France; ¶¶Nephrology Unit, Clinique duVert Galant, Tremblay-en-France, France; ***Nephrology Unit, Centre hospitalier de Saintonge, Saintes, France; †††Departement ofPediatrics, Centre Hospitalier Universitaire de Grenoble, Grenoble, France; ‡‡‡Departement of Pediatrics and ‡‡‡‡Departement ofNephrology, Centre de Référence des Maladies Rénales Rares du Sud-Ouest, Hôpital de Toulouse, Université Paul Sabatier, Toulouse,France; §§§Pediatric Nephrology Unit, Hôpital de la Timone, Assistance Publique des Hôpitaux de Marseille, Marseille, France; ǁǁǁPediatricNephrology Unit and *****Department of Nephrology, Hôpital Necker-Enfants-malades, Assistance Publique des Hôpitaux de Paris, Paris,France; ¶¶¶Nephrology Unit, Centre Hospitalier du Pays d’Aix, Aix-en-Provence, France; ****Pediatric Nephrology Unit, Hôpital Trousseau,Assistance Publique des Hôpitaux de Paris, Paris, France; §§§§Pediatric Nephrology Unit, Centre Hospitalier Universitaire de Montpellier,Montpellier, France; ǁǁǁǁFaculté de Médecine, Centre de Référence des Maladies Rénales Rares du Sud-Ouest, Aix-MarseilleUniversité–Vascular Research Center of Marseille, Marseille, France; ¶¶¶¶Nephrology Unit, Hôpital de la Conception, Assistance Publique desHopitaux de Marseille, Marseille,France; †††††Departement of Pediatrics, Hôpital Nord, Centre Hospitalier Universitaire de Saint Etienne,Saint Etienne, France; ‡‡‡‡‡Department of Pediatrics and Adolescent Medicine, Centre Hospitalier Universitaire d’Amiens, Amiens, France;§§§§§Service de Néphrologie Pédiatrique, Groupement Hospitalier Pellegrin, Centre Hospitalier Universitaire de Bordeaux, Centre deRéférence des Maladies Rénales Rares du Sud-Ouest, Bordeaux, France; ǁǁǁǁǁDepartment of Pediatrics, Centre Hospitalier UniversitaireCharles Nicolle, Rouen, France; ¶¶¶¶¶Nephrology Unit,Centre Hospitalier Universitaire Tours, Tours, France; ******Department of Pediatrics,Centre Hospitalier Pierre Oudot de Bourgoin-Jallieu, Bourgoin-Jallieu, France; ††††††Molecular Genetics Unit, Hospital do Divino EspíritoSanto de Ponta Delgada, Entidade Pública Empresarial Regional, Açores, Portugal; ‡‡‡‡‡‡Nephrology Unit, Centre Hospitalier Universitairede Dijon, Dijon, France; §§§§§§Pediatric Nephrology Unit, Centre Hospitalier Universitaire de Besançon, Besançon, France; ǁǁǁǁǁǁPediatricNephrology Unit, Hôpital Jeanne de Flandre, Centre Hospitalier Universitaire de Lille, Lille, France; ¶¶¶¶¶¶Pediatric Nephrology Unit,Centre Hospitalier Universitaire de Nantes, Nantes, France; *******Pediatric Nephrology Unit, Hôpitaux de Brabois, Centre HospitalierUniversitaire de Nancy, Vandoeuvre Les Nancy, France; †††††††Centre for Nephrology, University College London, London, UK; and‡‡‡‡‡‡‡Institut National de la Santé et la Recherche Médicale, Unité Mixte de Recherche en Santé 970, Paris-Cardiovascular ResearchCenter, Paris, France



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