Thyroid and hydrogen peroxide (H2O2): Duox1 and

Duox2 as novel actors in the thyroid pathophysiology

Xavier De Deken

IRIBHM

Université Libre de Bruxelles (U.L.B)

Brussels, Belgium

De Deken X.

29/05/2010

• Part 1 : Duox H2O2 generators: Tonic control of the

thyroid hormone synthesis

• Part 2 : Duox2 deficiency: New cause of congenital

hypothyroidism

De Deken X.

29/05/2010

• Part 1 : Duox H2O2 generators: Tonic control of the

thyroid hormone synthesis

• Part 2 : Duox2 deficiency: New cause of congenital

hypothyroidism

De Deken X.

29/05/2010

Thyroid hormone synthesis

Apical Pole

Basal Pole NIS

Na+/K+

ATPase

I-

Na+

Na+

K+

K+

O2H2O2

TgTgI

T4 + T3

NADPHNADP+ + H+

Pentose Cycle

I-

I-

I-

TPO

e-

De Deken X.

29/05/2010

The thyroid H2O2 generating system

• 1971 : Measurement of thyroid H2O2 (Bénard, Brault) / NADPH-dependent (Dumont)

• 1981 : Cyto-localization of H2O2 at the apex of thyrocytes (Björkman, Van sande)

• 1984 : Calcium-dependent H2O2 system (Virion)

• 1985 : NADPH oxidase complex (Nakamura)

• 1989 : Primary product is H2O2 not superoxide (Dupuy)

• 1994 : FAD containing complex (diphenyleneiodonium-sensitive) (Dupuy)

• 1999 : Purification and cloning from pig thyroid particles of p138Tox (partial Duox2sequence) (Dupuy)

• 2000 : Screening of human thyroid cDNA libraries with the Nox2 cDNA: ThyroidOxidases ThOX1 and ThOX2 (De Deken)

• 2001 : Human Genome Organization International Committee : Dual Oxidase - Duox

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29/05/2010

The NADPH oxidase family (1999-2001)

Nox1-1999 Colon, Prostate Cell proliferation ?

Uterus Proton channel ?

Nox2-1986 Phagocytes Immune defense

Nox3-2001 Inner ear Otoconia formation

Fetal kidney Embryogenesis ?

Nox4-2000 Kidney, skin Oxygen sensor ?

Osteoclast Bone resorption ?

Endothelial cells Vascular tone ?

Nox5-2001 Spleen Immune system ?

Testis Fertilization ?

Duox1-2000 Thyroid Iodide organification

Lung Host defense

Duox2-2000 Thyroid Iodide organification

Digestive tract Host defense

Tissue Function

Duox are not thyroid specific marker !!

De Deken X.

29/05/2010

Regulation of thyroid hormone synthesis

• TPO activity is dependent on substrates availability:

Iodide concentrated by NIS

Tg secreted in the follicular lumen

H2O2 generated by Duox

• Under sufficient iodide supply, H2O2 availability constitute the limiting step for thyroid hormone synthesis (Corvilain, 1991)

• TSH increase expression of NIS, TPO and TG but not DUOX

• TPO activity not directly regulated by TSH through post-translational modifications

Duox enzymatic activity must be acutely regulated

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29/05/2010

2006 - Identification of the Duox maturation factors: DuoxA1 / DuoxA2

Outside

Inside

DUOX1DUOX2

35.000 bp21.500 bp 4.000 bp

341134

DUOXA2 DUOXA1

6 8

12.000 bp

Chromosome: 15q15.3

From 2000: No functional reconstitution of

Duox activity in non-thyroid cells

Additional factor absolutely required

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29/05/2010

DuoxA-based functional assay (Rigutto, 2007)

Specific activity of Duox enzyme: H2O2 production normalized to Cos-7 cell surface

expression of the Duox proteins

Duox + DuoxA cDNA

Cellular transfection :

FACS analysis of cells :

H2O2 quantification in the incubation medium :

Fluorimetric

measurement

Protein expression by the cells

Stimulation by different agents

De Deken X.

29/05/2010

Thyroid hormone synthesis

Apical Pole

Basal Pole

IP3

DAG

Ca2+

NIS

Na+/K+

ATPase

I-

Na+

Na+

K+

K+

O2H2O2

TgTgI

T4 + T3

NADPHNADP+ + H+

Pentose Cycle

+

+

I-

I-

I-

TPO

e-

cAMP

Forskolin

PMA

Ionomycin

De Deken X.

29/05/2010

Calcium regulation

• Absence of Ca2+ in the reaction buffer No H2O2 production

• Mutation of Glu to Gln at position 12 of EF-Hand abolish the H2O2 generation:

I=1µM Ionomycin (Ca2+ ionophore)

Duox1 and Duox2 activities are Ca2+-dependent

De Deken X.

29/05/2010

PMA – PKC regulation : Duox2

• Dose/response with increasing PMA concentrations:

• Phosphorylation status on Duox2 with 32P Labeling:

250

150

C F 10 20

100

nM30

10

nM

1

nMDA2

PMA 5µM

32P

Anti-

Duoxs

Duox2 is activated by PMA – EC50=0.8nM

PMA increase phosphorylation of Duox2

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29/05/2010

cAMP-dependent activation of Duox1

H2O2 production of Duox/DuoxA cells stimulated with 1µM ionomycin, 1µM forskolin, 50µM 6MB-

cAMP or transfected with PKA catalytic subunit expressing plasmid :

Duox1 activity is stimulated by PKA activation with an EC50=0.1µM Fsk

Duox2 activity is not modulated by cAMP

De Deken X.

29/05/2010

PKA-mediated Duox1 phosphorylation

Analysis of Duox1 phosphorylation using anti-phospho-PKA substrate antibody (RXXS/T) :

Constitutive phosphorylation of the mature form of Duox1

Phosphorylation increased by cAMP-dependent protein kinase

De Deken X.

29/05/2010

PKA-mediated Duox1 Phosphorylations

Motif scanning [RK](2)-x-[ST] identify 3 potential PKA phospho-residues in Duox1 :

S955 – T1007 – S1217

R-R-A-S

955

K-K-V-T

1007

R-R-R-S

1217

S955 residue phosphorylated by PKA

S955 necessary for Duox1 response to cAMPactivation

De Deken X.

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Thyroid H2O2: Duox1 or Duox2 ?

• Thyroid gland: Organ expressing the highest amount of Duox1 and Duox2 mRNA (De Deken, 2000)

• Are both proteins expressed ? YES

Duox specific antibodies (U. Knaus, 2009)

• Are both enzymes functional ? yes

cAMP (Duox1) and DAG (Duox2)

stimulate H2O2 generation associated

with Duox phosphorylation (Rigutto, 2009)

But: Ca2+ necessary

Rat thyroid cell line, Pccl3, Duox1 is the

main H2O2 generator

- +TSH 4d

Duox1

TPO

Duox2

150kDa

100kDa

De Deken X.

29/05/2010

Thyroid H2O2: Duox1 or Duox2 ?

• Duox2 mRNA 2-5x > Duox1 (Pachucki, 2004)

• PMA responsiveness of Duox2 in nanomolar range: Constitutive tonic H2O2

generation (Rigutto, 2009)

Duox2 is the principal H2O2 generator

• If TSH blood levels increased: Duox activity stimulated mainly via Ca2+ + phosphorylations cAMP/DAG-dependent

Tight regulation to avoid oxidative stress

• Duox1: Emergency program revealed in case of Duox2 deficiency

De Deken X.

29/05/2010

• Part 1 : Thyroid hormone synthesis: Implication of the

Duox H2O2 generators

• Part 2 : Duox2 deficiency: New cause of congenital

hypothyroidism

De Deken X.

29/05/2010

Alteration of the thyroid metabolism

Hypothyroidism Hyperthyroidism

Iodide deficiency

High iodide intake (Wolff-Chaikoffeffect)

Auto-immune diseases (Hashimoto'sthyroiditis)

Anti-thyroid drugs

Pituitary (TSH) or hypothalamus (TRH)defect

…

Auto-immune diseases (Grave’s)

Toxic thyroid adenoma (Hot nodule)

Toxic multinodular goiter

Pregnancy (hCG TSH-like effect)

…

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Congenital hypothyroidism

• 1/4.000 newborns

• 80% dysembryogenesis – 20% dyshormonogenesis

• IOD (iodide organification defect): TPO / NIS / Tg

• Perchlorate discharge test: >10% and <90% PIOD // >90% TIOD

0

5

10

15

20

25

0 30 60 90 120 150 180

time (min)

12

3 I

upta

ke

(%)

normal

Patient 1

Patient 2

Patient 3

Patient 4

TIOD

NaClO4

(Moreno, 2001)

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29/05/2010

DUOX2 mutations associated with congenital hypothyroidism

• From 2002: 27 mutations found in ± 30 patients

• Only one homozygous mutation (R434X) causing TIOD (Moreno, 2002)

• 11 mutations in ECD – 11 mutations in first intracellular loop – 1 in catalytic domain

• Before 2008: Bi-allelic defect : Permanent CH - Mono-allelic defect : Transient CH

De Deken X.

29/05/2010

DUOX2 defect in Italy (Vigone et al. 2005)

Proband

Neonatal Diagnosis

Age (Days)

TSH (0.5-8.7 mU/L)

FT4 (1.5-2.4 ng/dL)

Thyroid Ab (anti-TPO, TRAb)

Ultrasonography

Urinary iodine (100-400 µg/L)

Treatment (L-T4)

7

173.2

0.5

Neg

Enlarged

------

+

Age (Days)

TSH (mU/L)

Urinary iodine (µg/L)

Treatment (L-T4)

-------

-------

-------

-------

Reevaluation (1 month without treatment)

Age (Years)

TSH (0.25-5.0 mU/L)

FT4 (0.7-1.7 ng/dL)

Urinary iodine (µg/L)

ClO4- discharge test (<10%)

4

6.3

1.2

139

28

Genotype

Allele 1

Allele 2

p.R376W

p.R842X

368 470380 390 400 410 420 430 440 450 460(368)

QALRVCNNYWIRE---------------------NPNLNSTQEVNELLLGMASQISELEDNIVVEDLRDYWPGPGKFSRTDYVASSIQRGRDMGLPSYSQALLThOX2_h (365)

PALRVCNSYWIRE---------------------NPNLNSAEAVNQLLLGMASQISELEDWIVVEDLRDYWPGPGKFSRTDYVASSIQRGRDMGLPSYTQALQDUOX2_p (365)

PALRVCNNYWIRE---------------------NPSLKTAQDVDQLLLGMASQISELEDRIVIEDLRDYWPGPDRYSRTDYVASSIQSGRDMGLPSYSQALQDUOX2_r (365)

PALRVCNSYWIRE---------------------NPNLKTAQDVDQLLLGMASQISELEDRIVIEDLRDYWPGPERFSRTDYVASSIQRGRDMGLPSYSQALLDUOX2_m (365)

PAMRLCNSYWSRE----------------------SIEMQQEDVDDLLLGMSSQIAEREDSIVVEDLQDYWYGPLKYSRADYVASWLQRGRDLGLPTYNQAREDUOX2_ch (353)

PALRVCNSYWLRE---------------------NANLNSAQAVDQLLLGMASQISELEDRIVVEDLRDYWPGSGKFSRTDYVASSIQRGRDMGLPSYTQALMThOX2_d (131)

RALRVCNSYWSRE---------------------HPSLQSAEDVDALLLGMASQIAEREDHVLVEDVRDFWPGPLKFSRTDHLASCLQRGRDLGLPSYTKARAThOX1_h (359)

RALRVCNSYWSRE---------------------HPNLQRAEDVDALLLGMASQIAEREDHMVVEDVQDFWPGPLKFSRTDHLASCLQRGRDLGLPSYTKARADUOX1_p (359)

GALRVCNSYWSRE---------------------NPKLQRAEDVDALLLGMASQIAEREDHLVVEDVQDFWPGPLKFSRTDYLASCLQRGRDLGLPSYTKAREDUOX1_r (359)

GALRVCNSYWSREREPGAQTLVLLAMSVFSPLLKHPKLQRAEDVDALLLGMASQIAEREDHVVVEDMQDFWPGPLKFSRTDYLASCLQRGRDLGLPSYTKAREDUOX1_m (359)

RALRVCNSYWSRK---------------------HPNLRRAEDVDALLLGMASQIAEREDHVVVEDVLDFWPGSLKFSRTDHVAGCLQRGRDLGLPSYTKARAThOX1_d (359)

-AVRLCSTWWDSS-----------------------GFFADTSVEEVLMGLASQISEREDPVLCSDVRDKLFGPMEFTRRDLGALNIMRGRDNGLPDYNTAREDUOX-dr (334)

PALRLCQNWWNAQ-----------------------DIVKEYSVDEIILGMASQIAERDDNIVVEDLRDYIFGPMHFSRLDVVASSIMRGRDNGVPPYNELRRDUOX_ce (356)

ALRVCNSYW RE P LQ AEDVD LLLGMASQIAERED IVVEDLRDYWPGPLKFSRTDYVAS IQRGRDLGLPSYT AR Consensus (368)

Permanent congenital hypothyroidism with

PIOD

Mutant protein inactive

in functional assay

De Deken X.

29/05/2010

DUOX2 defect in Italy (Vigone et al. 2005)

Proband Brother

Neonatal Diagnosis

Age (Days)

TSH (0.5-8.7 mU/L)

FT4 (1.5-2.4 ng/dL)

Thyroid Ab (anti-TPO, TRAb)

Ultrasonography

Urinary iodine (100-400 µg/L)

Treatment (L-T4)

7

173.2

0.5

Neg

Enlarged

------

+

11

9.6

1.8

Neg

Normal

550

-------

Age (Days)

TSH (mU/L)

Urinary iodine (µg/L)

Treatment (L-T4)

-------

-------

-------

-------

45

18.4

350

+

Reevaluation (1 month without treatment)

Age (Years)

TSH (0.25-5.0 mU/L)

FT4 (0.7-1.7 ng/dL)

Urinary iodine (µg/L)

ClO4- discharge test (<10%)

4

6.3

1.2

139

28

4

5.6

1.4

181

12

Genotype

Allele 1

Allele 2

p.R376W

p.R842X

p.R376W

p.R842X

368 470380 390 400 410 420 430 440 450 460(368)

QALRVCNNYWIRE---------------------NPNLNSTQEVNELLLGMASQISELEDNIVVEDLRDYWPGPGKFSRTDYVASSIQRGRDMGLPSYSQALLThOX2_h (365)

PALRVCNSYWIRE---------------------NPNLNSAEAVNQLLLGMASQISELEDWIVVEDLRDYWPGPGKFSRTDYVASSIQRGRDMGLPSYTQALQDUOX2_p (365)

PALRVCNNYWIRE---------------------NPSLKTAQDVDQLLLGMASQISELEDRIVIEDLRDYWPGPDRYSRTDYVASSIQSGRDMGLPSYSQALQDUOX2_r (365)

PALRVCNSYWIRE---------------------NPNLKTAQDVDQLLLGMASQISELEDRIVIEDLRDYWPGPERFSRTDYVASSIQRGRDMGLPSYSQALLDUOX2_m (365)

PAMRLCNSYWSRE----------------------SIEMQQEDVDDLLLGMSSQIAEREDSIVVEDLQDYWYGPLKYSRADYVASWLQRGRDLGLPTYNQAREDUOX2_ch (353)

PALRVCNSYWLRE---------------------NANLNSAQAVDQLLLGMASQISELEDRIVVEDLRDYWPGSGKFSRTDYVASSIQRGRDMGLPSYTQALMThOX2_d (131)

RALRVCNSYWSRE---------------------HPSLQSAEDVDALLLGMASQIAEREDHVLVEDVRDFWPGPLKFSRTDHLASCLQRGRDLGLPSYTKARAThOX1_h (359)

RALRVCNSYWSRE---------------------HPNLQRAEDVDALLLGMASQIAEREDHMVVEDVQDFWPGPLKFSRTDHLASCLQRGRDLGLPSYTKARADUOX1_p (359)

GALRVCNSYWSRE---------------------NPKLQRAEDVDALLLGMASQIAEREDHLVVEDVQDFWPGPLKFSRTDYLASCLQRGRDLGLPSYTKAREDUOX1_r (359)

GALRVCNSYWSREREPGAQTLVLLAMSVFSPLLKHPKLQRAEDVDALLLGMASQIAEREDHVVVEDMQDFWPGPLKFSRTDYLASCLQRGRDLGLPSYTKAREDUOX1_m (359)

RALRVCNSYWSRK---------------------HPNLRRAEDVDALLLGMASQIAEREDHVVVEDVLDFWPGSLKFSRTDHVAGCLQRGRDLGLPSYTKARAThOX1_d (359)

-AVRLCSTWWDSS-----------------------GFFADTSVEEVLMGLASQISEREDPVLCSDVRDKLFGPMEFTRRDLGALNIMRGRDNGLPDYNTAREDUOX-dr (334)

PALRLCQNWWNAQ-----------------------DIVKEYSVDEIILGMASQIAERDDNIVVEDLRDYIFGPMHFSRLDVVASSIMRGRDNGVPPYNELRRDUOX_ce (356)

ALRVCNSYW RE P LQ AEDVD LLLGMASQIAERED IVVEDLRDYWPGPLKFSRTDYVAS IQRGRDLGLPSYT AR Consensus (368)

Permanent congenital hypothyroidism with

PIOD

Phenotypic variability between the two sibling : Use of iodine-containing mouthwash during

pregnancy of the brother

Mutant protein inactive

in functional assay

De Deken X.

29/05/2010

DUOX2 defect in Argentina (Varela et al. 2006)

Case 1 Case 2

Reevaluation (4 weeks without treatment)

Age (Years)

TSH (0.25-5.0 mU/L)

Serum T4 (nmol/L)

Serum TG (µg/L)

ClO4- discharge test (<10%)

Treatment (L-T4)

5

156

37.3

431

46

+

4.5

>100

<12.87

131

60

+

Genotype

Allele 1

Allele 2

p.Q36H

p.S965fsX30

p.G418fsX65

p.R885fsX3

30 13240 50 60 70 80 90 100 110 120(30)

SLPWEVQRYDGWFNNLRHHERGAVGCRLQRRVPANYADGVYQALEEPQLPNPRRLSNAATRGIAGLPSLHNRTVLGVFFGYHVLSDVVSVETPGCPAEFLNIRThOX2_h (30)

SLTWEVQRYDGWFNNLRQHEHGAAGSPLRRLVPANYADGVYQALGEPLLPNPRQLSHTTMRGPAGLRSIRNRTVLGVFFGYHVLSDLVSIEKPGCPAEFLNIHDUOX2_p (30)

SLPREVQRYDGWFNNLKYHQRGAAGSQLRRLVPANYADGVYQALQEPLLPNARLLSDAVSKGKAGLPSAHNRTVLGLFFGYHVLSDLVSVETPGCPAEFLNIYDUOX2_r (30)

SLPWEVQRYDGWFNNLKYHQRGAAGSRLRRLIPANYADGVYQALEEPLLPNPRRLSDAVAKGKAGLPSVHNRTVLGVFFGYHVLSDLVSVETPGCPAEFLNIYDUOX2_m (30)

NITWEVQRYDGWYNNLQHRSRGSVGSRLLRLLPANYADGVYQALQEPHVPNARQLSNAVARGPSGLPSKRNTTVLAVFFGFHVLSDILGTEKPGCPAEFLNIHDUOX2_ch (19)

-------------------------------------------------------------------------------------------------------ThOX2_d (1)

PISWEVQRFDGWYNNLMEHRWGSKGSRLQRLVPASYADGVYQPLGEPHLPNPRDLSNTISRGPAGLASLRNRTVLGVFFGYHVLSDLVSVETPGCPAEFLNIRThOX1_h (24)

SISWEVQRFDGWYNNLMEHKWGSKGSRLQRLVPASYADGVYQPLGEPHLPNPRDLSNTAMRGPAGQASLRNRTVLGVFFGYHVLSDLVSIEKPGCPAEFLNIHDUOX1_p (24)

PVSWEVQRFDGWYNNLMEHRWGSKGSRLQRLVPASYADGVYQPLREPYLPNPRHLSNRVMRGPAGQPSLRNRTVLGVFFGYHVLSDLVSVETPGCPAEFLNIYDUOX1_r (24)

SISWEVQRFDGWYNNLMEHRWGSKGSRLQRLVPASYADGVYQPLKEPYLPNPRHLSNRVMRGSAGQPSLRNRTVLGVFFGYHVLSDLVSVETPGCPAEFLNIYDUOX1_m (24)

PISWEVQRFDGWYNNLMEHKWGSKGSRLQRLVPASYADGVYQPLGEPHLPNPRDLSNAAMRGPAGQASLRNRTVLGVFFGYHVLSDLVSVETPGCPAEFLNIRThOX1_d (24)

YSQTEKQRYDGWYNNLAHPDWGSVDSHLVRKAPPSYSDGVYAMAGAN-RPSTRRLSRLFMRGKDGLGSKFNRTALLAFFGQLVANEIVMASESGCPIEMHRIEDUOX-dr (2)

QQNEEFQRYDGWYNNLANSEWGSAGSRLHRDARSYYSDGVYSVNNSL--PSARELSDILFKGESGIPNTRGCTTLLAFFSQVVAHEIMQSNGVSCPLETLKIQDUOX_ce (23)

ISWEVQRYDGWYNNL H WGS GSRL RLVPA YADGVYQ L EP LPNPR LSN V RG AGLPSLRNRTVLGVFFGYHVLSDLVSVETPGCPAEFLNIYConsensus (30)

Permanent congenital hypothyroidism with PIOD

Mutant protein inactive

in functional assay

De Deken X.

29/05/2010

DUOX2 defect in Japan (Maruo et al. 2008)

Case 1

Neonatal Diagnosis

Age (Days)

TSH (0.1-4.3 mU/L)

FT4 (0.97-1.7 ng/dL)

Ultrasonography

15

95.4

0.43

Mild Enlargement

Reevaluation (stop treatment)

Age (Years)

TSH (0.25-5.0 mU/L)

FT4 (0.7-1.7 ng/dL)

11

2.16

1.35

Genotype

Allele 1

Allele 2

p.L479fsX3

p.K628fsX11

Transient mild congenital hypothyroidism

associated with complete DUOX2 inactivation

De Deken X.

29/05/2010

DUOX2 defect in Canada (Hoste et al. 2010)

Patient

Neonatal Diagnosis

Age (Days)

TSH (<5 mU/L)

FT4 (11-24 pmol/L)

Ultrasonography

31

23

3.9

Mild Enlargement

Reevaluation (stop treatment)

Age (Years)

TSH (0.1-6.2 mU/L)

FT4 (7.6-15.6 pmol/L)

18.5

2.65

8.5

Genotype

Allele 1

Allele 2

p.G1518S

p.G1172_F1548del

• First missense mutation associated with partial DUOX2 deletion

• First published inactivating mutation in the catalytic domain of DUOX2: G1518S

• Complete inactivation of DUOX2

• Transient congenital hypothyroidism

NADPH

De Deken X.

29/05/2010

DUOX2 defect in Canada (Hoste et al. 2010)

• G1518S mutant functionally inactive in reconstituted

system

• Correctly processed at the cell surface

25%

12%

WT

G1518S

De Deken X.

29/05/2010

Transient or permanent congenital hypothyroidism ?

• Genetic inter-population variability ?

• Partial compensation by Duox1 oxidase :

Inactivation of DUOX2 cause mainly a PIOD (29/30 cases)

Duox1 is functionally active in rat and human thyroid models (Rigutto, 2007 and

2009)

But: Duox1 mRNA 2-5x < than Duox2

• Environmental factors such as different iodine supply :

WHO: ± 2 billion in 2007 peoples concerned by iodide intake insufficient

? Deficiency in Italy and Argentina: Permanent CH

? High intake in Japan and Canada: Transient CH

Iodide: alternative treatment to L-T4 ?

De Deken X.

29/05/2010

Conclusions

Under sufficient iodide supply, H2O2 production is the limiting step for

thyroid hormone synthesis

T3/T4 synthesis controls :

Acute: Duox activity

Chronic: TPO, NIS, Tg gene expression

DUOX2 inactivating mutations quite frequent in Pt with CH and PIOD

(7Pt/20 - Persani, 2008) Treatment follow-up

Transient or persistent CH phenotype: not directly correlated to the number

of DUOX2 inactivated alleles

Homozygous DUOXA2 deficiency in Pt with mild CH (Zamproni, 2007)

Physiological role of Duox1 ?

De Deken X.

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ACKNOWLEDGMENTS

Montreal University :

G. van Vliet

IRIBHM – ULB :

J.E. Dumont and G. Vassart

F. Miot

B. Corvilain

S. Rigutto

C. Hoste

D. Wang

Y. Song

M. Milenkovic

J. Pachucki

C. Degraef

B. Bournonville

J. Van Sande

University of Chicago :

S. Refetoff

H. Grasberger

U.C.L :

M.C. Many

ARC