Gitelman and Bartter syndromes Rosa Vargas-Poussou Département de Génétique Hôpital Européen Georges Pompidou Paris

Gitelman and Bartter Syndromes Loosing salt tubulopathies Autosomal recessive inheritance Rare diseases; prevalence Gitelman Syndrome : 1/40.000 Bartter Syndrome : 1/100.000

Common characteristics Secondary activation of renin - angiotensin - aldosteron system Metabolic alkalosis Renal hypokalemia Normal or low blood pressure

Gitelman and Bartter Syndromes Na+ 7%

Cl Na+

Thiazides

Na

ClNa + Ca2+ Ca2+

Na+

Mg2+

K+

Na +

Ca2+ Mg2+ H+

+

Na+

Na+ 20% 2Cl Na+ K+

Furosemide

K+

K+ Ca++ Mg+

H+

Na+ Na+

+

NH4 (+) +

Vte

Na+ K+ Cl Barttin Cl K+

Gitelman Adolescent Adult Hypomagnesemi a Hypocalciuria Children Normo or hypercalciuria Polyuria Failure to thrive

Hydramnios Severe polyuria Hypercalciuria (-) Failure to thrive

HCO3-

Bartter

Antenatal Bartter

Gitelman and Bartter Syndromes Na+ 7%

Cl Na+

Thiazides

ClK+

Na+

Na+ Ca2+

Ca2+ Mg2+

Na+ 20%

Bartter type I (antenatal)

2Cl Na+ K+

Furosemide

Bartter type II (antenatal)

Ca2+

Na+

Mg2+

+

Na+

H+

Na

K+

K+ Ca++ Mg++ NH4+

(+)

H+

Na+

Na+ K+ Cl Barttin Cl K+

Gitelman Bartter type IV (antenatal with deafness)

HCO3-

Na+

Bartter (-)

Vte

Bartter type III (classic)

2Cl Na+ K+

Furosemide

Ca++ Mg++ NH4+

Na+ 7%

K+ H+

Na+ K+ Cl -

K+

Barttine

Cl K+

Na+ Na+

ClK+

Na+

H+

Na

Ca2+

Na+

Mg2+

+

Na+

Paracelline

(+)

(-) Vte

⊕

Na +

WNK1

K+ SGK WNK4

Na+ 2%

K+

Θ

Aldosteron

H2O RM

(-)

Hypokalem ic alkalosis

H2O (+) Cl-

H+ K+ H+

H+

Na+ Ca2+

Ca2+ Mg2+

HCO3-

ENaC

Na+ 20%

Cl Na+

Thiazides

HCO3 H2CO3 ACII CO2 H2O

-

HCO3-

2Cl Na+ K+

Furosemide

Ca++ Mg++ NH4+

Na+ 7%

K+ H+

Na+ K+ Cl -

K+

Barttine

Cl K+

Na+ Na+

ClK+

Na+

H+

Na

Ca2+

Na+

Mg2+

+

Na+

Paracelline

(+)

(-) Vte

⊕

Na +

WNK1

K+ SGK WNK4

Na+ 2%

K+

Θ

Aldosteron

H2O RM

(-)

Hypokalem ic alkalosis

H2O (+) Cl-

H+ K+ H+

H+

Na+ Ca2+

Ca2+ Mg2+

HCO3-

ENaC

Na+ 20%

Cl Na+

Thiazides

HCO3 H2CO3 ACII CO2 H2O

-

HCO3-

Gitelman Syndrome Thiazides Wnk-4

Cl Na+

ClK+

Na

Na+

+

Ca2+ Wnk-1

Population

-

Mg2+ H+

Ca2+

TRPV5 TRPM6 Na+

Na+

Ca2+

Na+

Mg2+

January 2001 to August 2009 448 patients with clinical diagnosis of GS (219 M and 229 F) French Network for Tubulopathies and Eunefron

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

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

SLC12A3 – Sequencing

MUTATIONS 172 different (100 novel)

448 PROBANDS 12%

6%

2%

14%

18% 52% 18% Compound heterozygous (n = 236) Homozygous (n = 79) One heterozygous mutation (n = 81) No mutation (n = 52)

70% (n=315)

15%

63% Missense (n=110)

Frameshift (n=26)

Splicing (n=24)

Nonsense (n=11)

Inframe (n=4)

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 dosage Peak Ratio Patien/control

< 0.7 deletion 0.7 - 1.3 Normal 1.3 – 2 duplication

77 patients • 53 of the 80 with one heterozygous point mutation • 24 of the 39 without mutations

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

– One exon deletion in 13 patients: • • • •

E9del, 1 patient E14del, 2 patients E18del, 1 patient E26del, 9 patients

– Two or more exon deletions or duplications in 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 1.

2. 22 19

20

1

23

2

21

3

B. QMPSF 1.

2.

LR - PCR 1

2

3

456

78

9 10 1112

13 14 15 16 17 18

19

20 21

22

E4_E6del

E2_E3del

23

24

25

26

E14del

2941bp 1786bp 4579bp

4082bp 2574bp

1872bp

Characterization of breakpoints 1

2 Intron1

Intron 3

Intron1

4

3 Intron 3

Intron 6

Intron17

2

Intron 13

Intron 14

Intron 23

Intron 25

6

5

1

Intron 3

3

456

78

1 (3342 bp) 2 (2720 bp) 3 (1508 bp)

Intron 18

9 10 11 12

13 14 15 16 17 18

19

20 21

4 (1183 bp) 5 (1355 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)

22

23

24

25

26

6 (10711 bp)

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 elements Cordaux R and Batzer MA, 2009

Non-Allelic Homologous Recombination

Human diseases

Belancio VP et al. Genome Research 2008 Non-Homologous End-Joining Large rearrangements Fork Stalling and Template 50 different genes Switching Wenli G et al. PathoGenetics 2008

Deletions Mechanism ?

Non allelic homologous recombination (NAHR) RepeatMasker http://www.repeatmaske r.org/

SLC12A3

1

2

3

456

78

1 (3342 bp) 2 (2720 bp) 3 (1508 bp)

9 10 1112

13 14 15 16 17 18

19

20 21

22

23

24

4 (1183 bp) 5 (1355 bp)

25

26

6 (10711 bp)

Alu(s) with homology > 80% 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

Non-allelic homologous recombination NAHR

1

2

3

456

78

9 10 1112

13 14 15 16 17 18

1 (3342 bp) 2 (2720 bp) 3 (1508 bp)

5’

TAAAGAGGCTTGAGGAAGACTTTTTCTTTCTTTTTTTTTTTT

4 patient TAAAGAGGCTTGAGGAAGACCCTGAGTGAGCTTCCAGGGCCT

3’

GAATCCCCTGTCCGAAGGACCCTGAGTGAGCTTCCAGGGCCT

19

20 21

22

23

24

25

4 (1183 bp) 5 (1355 bp)

5’

26

6 (10711 bp)

CTACTTGCTTATCACCGTGGCTCTGTGAGGACTGGGGACACAATCTG

6 patient CTACTTGCTTATCACCGTGGCTCTGAAGGCAGTAAAGTGGGGTGATG

3’

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)

GCTGTTGGGACTGTGGAGGGCTCTGAAGGCAGTAAAGTGGGGTGATG

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

Non-homologous end-joining NHEJ

26 25

26

10191pb

20000 10000 7000 5000 4000 3000 2000 1500 1000 700 500 400

AluJb

AluSx

AluSx AluSx AluSx AluSx AluJb AluJb

E26del in 9 patients

7 Intron 25

1

2

3

456

78

9 10 1112

(3342 bp)

2 3

13 14 15 16 17 18

4

19

20 21

(1183 bp)

5

(2720 bp)

3’UTR

25bp insertion

(1355 bp)

22

23

24

25

26

6

(10711bp)

7

(2416bp)

(1508 bp)

5’ GGGTAGGGCTTGTCCCAGGTGAAGCTTTGTGGATGGAACTTCCAAGTGTGACATAGCTGTTTAGTATCCCAGTTACCCTTCTCAGAGGAG

patient GGGTAGGGCTTGTCCCAGGTGAAGCTTTTTAGTAGAGATGGGGTTTAGTAGAGATTTTTAGTAGAGATGGGGTTTCACCATGTTGACCAG

3’

CTCCCGAATAGCTGGGATTACAGGCACCTGCCATCACACGAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGACCAG

a1.

Patient:E1_7del

1.

2.

7

G A

2 1

5

3

6

6

3 4 1 2

5

7

2. Mother: Nle

b

A G G A A A G C T G C T T C A A G G

A G A G G C A G C G C G T T A G G G

rs7202364 rs2043635 rs12444217 rs1529929 rs7199480 rs4784730 rs1347591 rs12445993 rs12443821 rs12932041 rs4784732 rs1436424 rs12920659 rs12599065 rs12921781 rs3829502 rs11640954 rs34136389

Log2 (ratio) 56,860 Mb

rs7202364 rs2043635 rs12444217 rs1529929 rs7199480 rs4784730 rs1347591 rs12445993 rs12443821 rs12932041 rs4784732 rs1436424 A rs12920659 A rs12599065 rs12921781 rs3829502 rs11640954 rs34136389

4

-4

-2

-1

0

+1

+2

+4

cent

56,899,150 bp

4 2 3

5

6

56,902,220 bp

56,904,630 bp 56,912,240 bp

tel

Ch. 16

56,950 Mb

rs7202364 rs2043635 rs12444217 rs1529929 rs7199480 rs4784730 rs1347591 rs12445993 rs12443821 rs12932041 rs4784732 rs1436424 rs12920659 rs12599065 rs12921781 rs3829502 rs11640954 rs34136389

SLC12A3

A A

A G G A A A G C T G C T T C A A G G

13090 bp 2410 bp

1

G A

NUP93

7

Gitelman syndrome - Molecular analysis

Sequencing + MLPA 448 PROBANDS

6% 2% 6% 8%

3%

13%

17%

55%

14%

59%

17% Compound heterozygous (n = 260) Homozygous (n = 79) One heterozygous mutation (n = 81) No mutation (n = 38) CLCNKB (n=14)

91% mutation detection rate

Missense (n=110) Frameshift (n=26) Splicing (n=24) Nonsens (n=11) Inframe (n=4) Large rearrangements (n=11) Vargas-Poussou R et al., JASN 2011

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

627 Probands 8%

Sequencing + MLPA

3%

6%2% 7%

15%

13% 57% 17%

13%

59%

Compound heterozygous (n = 341) Homozygous (n = 104) One heterozygous mutation (n = 90) No mutation (n = 72) CLCNKB (n=20

92% mutation detection rate

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

Gitelman syndrome Database January 2012

• 375 patients : 189 M and 186 F • 3 Centres

9.5%

– Paris : 314 – Brussels: 32 patients – Nijmegen: 29 patients 16%

• Mutations – SLC12A3: 363 – CLCNKB: 12

7%

n= 348

Med IQR: 25 (14-38)

Symptoms at diagnosis 25 20 15 % 10 5 0

Fortuitous cramps Asthenia Failure to Thrive Polyuria Tetany Arrhytmia Malaise Chondrocalcinosis Vomiting Paresia and paresthesia Hypotonia Abdominal pain Enuresis

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% 22 %

n= 322 Med IQR: 0.62 (0.52-0.68)

n= 151 Med IQR 0.08 (0.03-0.18)

Classic Bartter Syndrome Bartter syndrome type III

Na+ 2Cl K+

N = 92

Na+

K+

K+ Cl 21%

Cl K+

K+ H+

Na+

RSCa Na+

(+)

Ca++ Mg++ NH4+

21%

HCO326%

27%

Claudins 16 - 19

Alkalose et Hypochlorémie ++++

(-)

5%

Compound heterozygous Homozygous Heterozygous SLC12A3 No mutation

52%

CLCNKA-CLCNKB – 1p36

Classic Bartter/Gitelman Missense

17%

Nonsense 7% 48%

Frameshift

14%

ClC-kb channel : 687 aa.

Splicing

Large rearrangements

14%

N

Homozygous whole gene deletion

Classic Bartter

73

10

Gitelman

22

2

p=0.037

n=85, Med. IQR n=25, Med. IQR 30 (28-32) 32.9 (28.5-36)

p