Gitelman and Bartter syndromes - HÔPITAL · PDF fileGitelman and Bartter Syndromes Loosing salt tubulopathies Autosomal recessive inheritance Rare diseases; Common characteristics

Gitelman and Bartter syndromes

Rosa Vargas-PoussouDépartement de GénétiqueDépartement de Génétique

Hôpital Européen Georges PompidouParis

Gitelman and Bartter Syndromes

� Loosing salt tubulopathies

�Autosomal recessive inheritance

�Rare diseases;

�Common characteristics �Secondary activation

of renin - angiotensin - aldosteron system�Rare diseases;

prevalence�Gitelman Syndrome :

1/40.000�Bartter Syndrome :

1/100.000

- aldosteron system�Metabolic alkalosis�Renal hypokalemia�Normal or low blood

pressure

Na+

20%

Na+ 7%Thiazides

Ca2+

Na+Cl -

Na+ Ca2+

Na+

K+

Ca2+

Na+H+

Mg2+ Na+

Na+ Mg2+

Cl-

Gitelman and Bartter SyndromesGitelman

Bartter

AdolescentAdultHypomagnesemiaHypocalciuria

ChildrenNormo or hypercalciuria20%

Furosemide

(+)

Na+

K+

2Cl - Na+

K+

K+

Na+H+

K+

Cl -

Cl -

Na+HCO3

-

Ca++

Mg+

+

NH4+(-)

Vte

K+

Barttin

Antenatal Bartter

hypercalciuriaPolyuriaFailure to thrive

HydramniosSevere polyuriaHypercalciuriaFailure to thrive

Na+

20%

Na+ 7%Thiazides

Ca2+

Na+Cl -

Na+

Ca2+

Na+K+

Ca2+

Na+H+

Mg2+ Na+

Na+ Mg2+

Cl-

Bartter type I

Gitelman and Bartter Syndromes

Gitelman

Bartter type IV(antenatal with 20%

Furosemide

(+)

Na+

K+

2Cl - Na+

K+

K+

Na+H+

K+

Cl -

Cl -

Na+HCO3

-

Ca++

Mg++

NH4+

(-)Vte

K+

Barttin

Bartter type I(antenatal)

Bartter type II(antenatal)

Bartter type III(classic)

Bartter

(antenatal with deafness)

Na+

Na+ 7%

Thiazides

Ca2+

Na+Cl -

Na+

Ca2+

Na+K+

Ca2+

Na+H+

Mg2+ Na+

Na+ Mg2+

Cl-Furosemide Na+

K+

2Cl - Na+

K+

K+

Na+H+

K+

Cl -

Cl -

Na+HCO3

-Ca++

Mg++

NH4+

(-)(+)Vte

K+

Paracelline

Barttine

Na + K+

SGK

WNK1 ENaC

⊕

WNK4 Na+

20%

H2OH2O

K+

Aldosteron

SGK

RM

WNK4

ΘNa+

2%

(-) (+)

Cl-

HCO3-

K+

H+

H+

CO2 H2O

H2CO3

H+ HCO3-

ACII

Hypokalemic alkalosis

Na+

Na+ 7%

Thiazides

Ca2+

Na+Cl -

Na+

Ca2+

Na+K+

Ca2+

Na+H+

Mg2+ Na+

Na+ Mg2+

Cl-Furosemide Na+

K+

2Cl - Na+

K+

K+

Na+H+

K+

Cl -

Cl -

Na+HCO3

-Ca++

Mg++

NH4+

(-)(+)Vte

K+

Paracelline

Barttine

Na + K+

SGK

WNK1 ENaC

⊕

WNK4 Na+

20%

H2OH2O

K+

Aldosteron

SGK

RM

WNK4

ΘNa+

2%

(-) (+)

Cl-

HCO3-

K+

H+

H+

CO2 H2O

H2CO3

H+ HCO3-

ACII

Hypokalemic alkalosis

Gitelman Syndrome

Ca2+

Na+Cl -

Na+

Ca2+

Thiazides

Na+K+

Ca2+

Na+

Mg2+Na+

Na+ Mg2+

Cl-

Wnk-4

Wnk-1TRPV5

TRPM6

Population�January 2001 to August 2009 �448 patients with clinical diagnosis of GS �(219 M and 229 F) �French Network for Na+

H+

Reabsorption:Na+ 7%Ca2+ 10- 15%Mg2+ 10%

�French Network for Tubulopathies and Eunefron

Criteria �Renal hypokalemia�Metabolic alkalosis�NaCl losing�Secondary activation of the RAA system�Hypomagnesemia�Hypocalciuria

SLC12A3 – Sequencing

MUTATIONS 172 different (100 novel)

448 PROBANDS

63%15%

14%6% 2%

Missense (n=110) Frameshift (n=26)

Splicing (n=24) Nonsense (n=11)

Inframe (n=4)

52%

18%

18%

12%

Compound heterozygous (n = 236)

Homozygous (n = 79)

One heterozygous mutation (n = 81)

No mutation (n = 52)

70%(n=315)

SLC12A3 point mutations Frequency and distribution - 172 mutations in 711 alleles

Kunchaparty, S, et al. Am J Physiol, 1999.De Jong, JC, et al. J Am Soc Nephrol, 2002.

52 patients without mutations

�Analysis of the CLCNKB gene in 49 patients: detection of molecular abnormalities in 14

� 38 patients without molecular abnormalities (8.4%)

Search for heterozygous large rearrangements

�MLPA (Multiplex Ligation-dependant Probe Amplification)� Salsa® MLPA® kit P136 SLC12A3 Gitelman Syndrome MRC Holland

�QMPSF (Quantitative Multiplex PCR Short Fluorescent Fragments)

Semi-quantitative techniques: allele dosagePeak Ratio Patien/control

< 0.7 deletion0.7 - 1.3 Normal1.3 – 2 duplication

�77 patients• 53 of the 80 with one heterozygous point

mutation• 24 of the 39 without mutations

MLPA and QMPSF results

– One exon deletion in 13 patients:

– Two or more exon deletions or duplications in 11 patients

11 different large rearrangements in 24 out of 53 patients with one heterozygous point mutation tested

13 patients:• E9del, 1 patient• E14del, 2 patients• E18del, 1 patient• E26del, 9 patients

11 patients • E1_E7del, 2 patients• E2_E3del, 2 patients• E4_E6del, 3 patients• E19_E23del, 1 patient• E24_E25del, 1 patient• E1_E3dup, 1 patient• E1_E4dup, 1 patient

A. MLPA

19 20 2122 23

1 2

3

1. 2.

B. QMPSF1. 2.

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 1617 18 19 20 21 22 23 24 25 26

LR - PCR

E4_E6del E14delE2_E3del

4082bp

2574bp

2941bp1786bp

4579bp

1872bp

Characterization of breakpoints

Intron 13 Intron 14

Intron1 Intron 3

1 2

Intron1 Intron 3

5

4

6

3

Intron 3 Intron 6

Intron 23 Intron 25Intron17 Intron 18

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 1617 18 19 20 21 22 23 24 25 26

5 (1355 bp)2 (2720 bp)

1 (3342 bp) 4 (1183 bp) 6(10711 bp)

3 (1508 bp)

1. E2_E3 del: c.282+667_c.506-205del

2. E2_E3 del: c.283-273_c.506-213del

3. E4_E6 del: c.506-305_c.852+185del, (3 patients)

4. E14del: c.1169+773_c.1825+247del, (2 patients)

5. E18del: c.2178+269_c.2285+685del

6. E24_E25del: c.2748-324_c.2952-505

Mechanisms of large genomic rearrangements

Transposable elementsCordaux R and Batzer MA, 2009

Wenli G et al. PathoGenetics 2008

Non-Allelic Homologous Recombination

Non-Homologous End-Joining

Fork Stalling and TemplateSwitching

Human diseasesBelancio VP et al. Genome Research 2008

Large rearrangements 50 different genes

Deletions Mechanism ?

�Non allelic homologous recombination (NAHR)(NAHR)

�RepeatMasker http://www.repeatmasker.org/

�SLC12A3

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 1617 18 19 20 21 22 23 24 25 26

Alu(s) with homology > 80%

5 (1355 bp)2 (2720 bp)

1 (3342 bp) 4 (1183 bp) 6(10711 bp)

3 (1508 bp)

Alu(s) with homology > 80%

Non-allelic homologous recombinationNAHR

1. E2_E3 del: c.282+667_c.506-205del

2. E2_E3 del: c.283-273_c.506-213del



5. E18del: c.2178+269_c.2285+685del

6. E24_E25del: c.2748-324_c.2952-505

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 1617 18 19 20 21 22 23 24 25 26

5 (1355 bp)2 (2720 bp)

1 (3342 bp) 4 (1183 bp) 6(10711 bp)

3 (1508 bp)

CTACTTGCTTATCACCGTGGCTCTGTGAGGACTGGGGACACAATCTG

1. E2_E3 del: c.282+667_c.506-205del

2. E2_E3 del: c.283-273_c.506-213del



5. E18del: c.2178+269_c.2285+685del

6. E24_E25del: c.2748-324_c.2952-505

TAAAGAGGCTTGAGGAAGACTTTTTCTTTCTTTTTTTTTTTT

GAATCCCCTGTCCGAAGGACCCTGAGTGAGCTTCCAGGGCCT

TAAAGAGGCTTGAGGAAGACCCTGAGTGAGCTTCCAGGGCCT

5’

patient

3’

CTACTTGCTTATCACCGTGGCTCTGTGAGGACTGGGGACACAATCTG

CTACTTGCTTATCACCGTGGCTCTGAAGGCAGTAAAGTGGGGTGATG

GCTGTTGGGACTGTGGAGGGCTCTGAAGGCAGTAAAGTGGGGTGATG

5’

patient

3’

4 6

Non-homologous end-joining NHEJ

25 26

Alu

Sx

Alu

Sx

Alu

Sx

Alu

Sx

Alu

JbA

luJb

Alu

Sx

Alu

Jb

E26del in 9 patients

10191pb

26

20000100007000500040003000

2000

1500

1000

700500400

7

Intron 25 3’UTR25bp insertion

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 1617 18 19 20 21 22 23 24 25 26

5’

patient

3’

GGGTAGGGCTTGTCCCAGGTGAAGCTTTGTGGATGGAACTTCCAAGTGTGACATAGCTGTTTAGTATCCCAGTTACCCTTCTCAGAGGAG

GGGTAGGGCTTGTCCCAGGTGAAGCTTTTTAGTAGAGATGGGGTTTAGTAGAGATTTTTAGTAGAGATGGGGTTTCACCATGTTGACCAG

CTCCCGAATAGCTGGGATTACAGGCACCTGCCATCACACGAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGACCAG

(1355 bp)(2720 bp)

(1508 bp)

(3342 bp) (1183 bp)(10711bp)

(2416bp)�

2

3

4

5

6

7

0 +1 +2-1-2-4 +4

56,860 Mb

Log2 (ratio)

2.

1

2

3

4

5

7

6

a

1

2

3

4

5

7

6

a

61 2

3 4 5 761 2

3 4 5 761 2

3 4 5 7

bb

1.

2.

cent

A G GAAAG CTGC TTCAAGG

rs7202364rs2043635rs12444217rs1529929rs7199480rs4784730rs1347591rs12445993rs12443821rs12932041rs4784732rs1436424rs12920659rs12599065rs12921781rs3829502rs11640954rs34136389

AGAGGCAGCGCGTTAG GG

AA

GA


1.Patient:E1_7del

Mother: Nle

24

10

bp

13

09

0 b

p

56,950 Mb

56,899,150 bp

56,902,220 bp

56,904,630 bp

56,912,240 bp

1

2

3

4

5

7

6

b

1

2

3

4

5

7

6

b

tel

Ch. 16

NU

P93

SLC

12A3

A G GAAAG CTGC TTCAAGG


AA

GA

Mother: Nle

Gitelman syndrome - Molecular analysis

Sequencing + MLPA

6%2% 6% 448 PROBANDS

59%14%

13%

6%2% 6%

Missense (n=110)Frameshift (n=26)Splicing (n=24)Nonsens (n=11)Inframe (n=4)Large rearrangements (n=11)

55%

17%

17%

8% 3%


Homozygous (n = 79)



CLCNKB (n=14)

91% mutation detection rate

Vargas-Poussou R et al., JASN 2011

Gitelman syndrome - Molecular analysis of the Paris cohort January 2001 to December 2011

Sequencing + MLPA

2% 7%

627 Probands

15%8% 3%

59%13%

13%

6%2% 7%

Missense (n=129)Frameshift (n=29)Splicing (n=28)Nonsens (n=13)Inframe (n=4)Large rearrangements (n=15)

92% mutation detection rate

57%17%

15%


Homozygous (n = 104)



CLCNKB (n=20

Gitelman syndrome DatabaseJanuary 2012

• 375 patients : 189 M and 186 F

• 3 Centres– Paris : 314

9.5%

– Paris : 314– Brussels: 32

patients– Nijmegen: 29

patients

• Mutations – SLC12A3: 363– CLCNKB: 12

16%

n= 348 Med IQR: 25 (14-38)

7%

Symptoms at diagnosis

15

20

25FortuitouscrampsAstheniaFailure to ThrivePolyuriaTetany

0

5

10

15

%

TetanyArrhytmiaMalaiseChondrocalcinosisVomitingParesia and paresthesiaHypotoniaAbdominal painEnuresis

Biochemical data at diagnosis

n= 354 Med IQR: 2.70 (2.48-3.00) n= 280 Med IQR 30 (28-32) n= 235 Med IQR 98 (95-100)


n= 250 Med IQR: 138.8 (137-140) n= 280 Med IQR 2.37 (2.25-2.47) n= 41 Med IQR 286 (240-329)


39%

n= 322 Med IQR: 0.62 (0.52-0.68) n= 151 Med IQR 0.08 (0.03-0.18)

22 %

Classic Bartter SyndromeBartter syndrome type III

Na+

K+2Cl -

Na+K+

K+

Na+

K+

Cl -

Cl -

K+

RSCa

N = 92

21%21%

Na+H+

Na+HCO3

-

Ca++

Mg++

NH4+

(-)(+) Claudins 16 - 19

RSCa

Alkalose et Hypochlorémie ++++

26%

5%

27%

Compound heterozygousHomozygousHeterozygousSLC12A3No mutation

52%

Classic Bartter/Gitelman

17%Missense

Nonsense

CLCNKA-CLCNKB – 1p36

ClC-kb channel : 687 aa.

48%

14%

14%

7%Nonsense

Splicing

Frameshift

Largerearrangements

N Homozygous whole gene deletion

Classic Bartter 73 10

Gitelman 22 2

n=85, Med. IQR30 (28-32)

n=25, Med. IQR 32.9 (28.5-36)

n=61, Med. IQR 97 (94.5-99)

n=25, Med. IQR 89 (80-96.5)

p=0.037 p<0.0001

n=94, Med. IQR 17.5 (8-36)

n=35, Med. IQR 1 (0.4-3)

p<0.0001n=89, Med. IQR 0.59 (0.51-0.68)

n=26, Med. IQR0.87 (0.77-0.96)

n=81, Med. IQR 0.05 (0.03-0.11)

n=21, Med. IQR 0.62 (0.23-1.14)

p<0.0001

p<0.0001

Antenatal Bartter (n=97)

26%

11%

23%KCNJ1

SLC12A1

CLCNKB

BSND

No Mutation

20%20%

No Mutation

Bartter Type III CLCNKB mutations

25%

53%

22%

GitelmanClassic BartterAntenatal Barter

17%3%

Antenatal Barter Syndrome - Type IGene SLC12A1 – 15q15-21

831 2 3 4 5 7 8 9 10 11 126

COOH

NH2

Na-K 2-Cl : 1.099 aa. Gene: 26 exons

49%

24%

7%

17%

MissenseSplicingNonsenseFrameshiftInframe

N = 19 : 11 homozygous, 6 compound heterozygous

M1 M2

extracellulaire

H5

P

P

aa hydrophobe

proline

aa polaire

aa basique

aa acide

site PKA

site PKC

site de glycosilation

21%

Antenatal Barter Syndrome - Type IIGene KCNJ1 – 11q24-25

NH2

COOH

M0

intracellulaire

P

P

P

P

P

site Walker-A

D omaine de rˇgulation de la liaison l'ATP

K channel Kir 1.1. : 391 aa. Gene: 5 exons

72%

7%

MissenseNonsenseFrameshift

N = 26 : 11 homozygous, 14 compound heterozygous, 1 Heterozygous.

Transient Hyperkalemia

Antenatal Barter Syndrome - Type IIIGene CLCNKB – 1p36

ClC-kb channel. : 687 aa. Gene: 19 exons

Antenatal Bartter

Whole homozygous gene deletion (9/19)8 missense mutations3 frameshift or nonsense2 splicing

N = 19 : 11 homozygous, 5 compound heterozygous, 3 Heterozygous.

Barttin. : 320 aa.

N =11 9 homozygous2 Compound Heterozygous.

Antenatal Barter Syndrome with deafness - Type IVBSND – 1p31

Bartter IVa

CLCNKA-CLCNKB – 1p36

Digenic Inheritance

Bartter IVb

aa. Bartter IVa

Rickheit G et al EMBO 2008

Antenatal Bartter SyndromePhenotype -Genotype correlation

Brochard, K. et al. Nephrol. Dial. Transplant. 2009 24:1455-64

KCNJ1

SLC12A1 CLCNKB

BSND

Nombre 23 17 6 6

Médiane 25 26 28 22

KCNJ1

SLC12A1 CLCNKB

BSND

Nombre 31 28 16 7

Médiane 32 32 37 31,8

* p = 0,021 ** p = 0,002 *** p = 0,001

Antenatal Bartter Syndrome

KCNJ1

SLC12A1 CLCNKB

BSND

Nombre 32 18 10 7

Médiane 1321,5

131 131 121

KCNJ1

SLC12A1 CLCNKB

BSND

Nombre 28 21 14 9

Médiane 5 3 2.6 2.4

* p = 0,002 ** p = 0,0001 *** p = 0,0006

Antenatal Bartter Syndrome

KCNJ1

SLC12A1 CLCNKB

BSND

Nombre 18 15 9 7

Médiane 26.5 28 39 27

KCNJ1

SLC12A1 CLCNKB

BSND

Nombre 17 10 7 6

Médiane 92 89 76 83

* p = 0,01* p = 0,01 ** p = 0,002***p=0,003

EAST : Epilepsy, Ataxia, Sensorineural Deafness,Tubulopathy

Cl -Thiazides Cl-

Reabsorption:Na+ 7%Ca2+ 10- 15%Mg2+ 10%

Scholl UI et al. PNAS 106, 2009

Bockenhauer D et al. New Engl J Med 360, 2009

SeSAME: Seizures, sensorineural deafness,

ataxia, mental retardation, electrolyte imbalanceCa2+

Na+Cl

Na+

Mg2+

Thiazides

Na+K+

Na+

H+

Mg2+

Na+ Mg2+

Cl-

Wnk-4

Wnk-1TRPV5

TRPM6

K+

KCNJ10, 1q23.2K channel kir 4.1

Heterodimer with kir 5.1

• ♀, Born 02/2010.

• First child, consanguineous family

• 6 months: failure to thrive, hypotonia.

• Biochemical data:– Metabolic alkalosis

D12826 130D1S436 204D1S2644 245

CLCNKB

130 204245

SLC12A3D16S3039 248 D16S3057 194D16S3071 89

260 20091

D12826 130D1S436 204D1S2644 245

CLCNKB

130 204245

SLC12A3D16S3039 248 D16S3057 194D16S3071 89

260 20091

CLCNKB analysis: No mutation

– Metabolic alkalosis• pH: 7.52• K : 2.6 mmol/L

– Renal hypokalemia• Ur K: 82mmol/L

– Hypocalciuria• Ca/creat :

0.035mmol/mmol)

• Age of one year:– Seizures– Neurological evaluation

• Ataxia, nistagmus• Developmental

retardation

– ORL evaluation• Deafness

KCNJ10 gene analysis: known homozygous mutation, p.Arg175Gln

Antenatal Bartter• Congenital Chloride diarrhoea

� Autosomal recessive inheritance� SLC26A3 - 7q31.1� Faecal Cl > 90 mmol/L

Differential Diagnosis

Classic Bartter/Gitelman

• Cystic Fibrosis– Autosomal recessive inheritance– CFTR - 7q31.2

• Autosomal Dominant hypocalcemia

– Bartter type V

• Diuretics toxicity• Eating disorders

• Other causes of hypokalemia– Periodic paralysis with

hypokalemia

• Other causes of hypomagnesemia

– Proton pump inhibitors (Inexium– Mody 5

• Pseudophypoaldosteronism 1– Autosomal recessive

– SCNN1A, SCNN1B, SCNN1G

– Autosomal dominat• NR3C2

– Persistent hyperkalemia

Wedenoja S et al. Aliment Pharmacol Ther. 2010, Human Mut 2011

Parameter Antenatal Bartter Classic Bartter Gitelman

Type I Type II Type IV Type III

Age of presentation In utero In utero In utero In utero Infant- toddler, preschool or school

children

Adolescent/adult /children

Polyhydramnios/ Prematurity

+++ +++ +++ ++ - -

Failure to thrive +++ +++ +++ +++ ++ +

ConclusionMain biological and clinical characteristics and genetic classification

Failure to thrive +++ +++ +++ +++ ++ +

Deafness - - Yes - - - Polyuria +++ +++ +++ +++ ++ -

Tetany crisis - - - - Occasional Common

Magnesemia Normal Normal Normal Normal Normal or low Low (sometimes normal)

Transient Hyperkalemia

- +++ - - - -

Hypochloremic alkalosis

++ ++ +++ ++++

++

Calciuria High +++ High +++ Normal or High +

Normal or High + Low or normal

Nephrocalcinosis +++ +++ ++ +/- - Urinary

prostaglandins

High +++ High +++ High +++ High +++ Normal or High + Normal

Indomethacin response

+++ +++ +++ +++ ++ +

Conclusions and perspectives

• Gitelman syndrome treatment– National “PHRC” GITAB : Dr Anne Blanchard

• Database analysis : – Phenotype-genotype correlation– Phenotype-genotype correlation– Subgroup analysis, i.e. older or younger patients– Follow-up analysis : long term morbidity

• Cardiovascular• Rheumatologic• Ocular

• Analysis of heterozygous relatives• New genes ?

Thanks

� Paris � EGPH Genetic

Department and Clinical Research Center

� Brussels� Pr Olivier

Devuyst� Pr Karin Dahan

� Patients and their families

Research Center� Pr Xavier Jeunemaitre� Dr Anne Blanchard� Technicians� Diana Kahila

� Physicians� Tubulopathies French

Network

� Pr Karin Dahan

� Nijmegen� Pr Nine Knoers� Physicians

Documents

Gitelman and Bartter syndromes - HÔPITAL · PDF fileGitelman and Bartter Syndromes Loosing salt tubulopathies Autosomal recessive inheritance Rare diseases; Common characteristics