thyroid and hydrogen peroxide (h2o2): duox1 and duox2 as ...thyroid and hydrogen peroxide (h 2 o 2):...
TRANSCRIPT
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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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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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
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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
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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.
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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
De Deken X.
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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.
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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.
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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
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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.
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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)
De Deken X.
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.
29/05/2010
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