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
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
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
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
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
rs7202364rs2043635rs12444217rs1529929rs7199480rs4784730rs1347591rs12445993rs12443821rs12932041rs4784732rs1436424rs12920659rs12599065rs12921781rs3829502rs11640954rs34136389
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
rs7202364rs2043635rs12444217rs1529929rs7199480rs4784730rs1347591rs12445993rs12443821rs12932041rs4784732rs1436424rs12920659rs12599065rs12921781rs3829502rs11640954rs34136389
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%
Compound heterozygous (n = 260)
Homozygous (n = 79)
One heterozygous mutation (n = 81)
No mutation (n = 38)
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%
Compound heterozygous (n = 341)
Homozygous (n = 104)
One heterozygous mutation (n = 90)
No mutation (n = 72)
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)
Biochemical data at diagnosis
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)
Biochemical data at diagnosis
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
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