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The Journal of Clinical Endocrinology & Metabolism 86(10):4843– 4848
Copyright © 2001 by The Endocrine Society
Goiter and Hypothyroidism in Two Siblings due to
Impaired Caⴙ2/NAD(P)H-Dependent
H2O2-Generating Activity
MARCIA D. L. FIGUEIREDO, LUCIENE C. CARDOSO, ANDREA C. F. FERREIRA,
DENISE V. B. CAMPOS, MANOEL DA CRUZ DOMINGOS, ROSSANA CORBO,
LUIZ EURICO NASCIUTTI, MARIO VAISMAN, AND DENISE P. CARVALHO
Instituto de Biofı́sica Carlos Chagas Filho (M.D.L.F., L.C.C., A.C.F.F., D.V.B.C., D.P.C.), Departamento de Histologia e
Embriologia (L.E.N.), and Serviços de Endocrinologia (M.V.), Cirurgia (M.d.C.D.), and Medicina Nuclear (R.C.), Hospital
Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, Brazil
We report herein the study of two siblings (DESM and DSM)
with hypothyroidism, goiter, and positive perchlorate discharge tests (50% and 70%) in a family (M) with no history of
consanguinity. Thyroid gland histology showed a predominance of hyperactive follicles, with high epithelial cells and
variable colloid content. Thyroid peroxidase iodide oxidation
(DESM, 1034; DSM, 1064 U/g protein) and albumin iodination
(DESM, 16; DSM, 8 nmol I/mg protein) activities were within
the normal range. Tg content was normal in both glands compared with that in diffuse toxic goiter (DESM, 28; DSM, 17;
diffuse toxic goiter, 19 mg/g tissue), and Tg could be normally
iodinated by thyroid peroxidase in vitro (DESM, 3.4; DSM, 4.3;
diffuse toxic goiter, 6.3 nmol I/mg Tg). Thyroid cytochrome c
reductase activities in these goiters were higher than that in
paranodular tissues (DESM, 473; DSM, 567; paranodular tissues, 78 nmol NADPⴙ/h/mg protein). However, thyroid NADPH
oxidase activities were very low both in the particulate 3,000 ⴛ
g (DESM, 4.8; DSM, 44; paranodular tissues, 224 nmol H2O2/
h/mg protein) and in the particulate 100,000 ⴛ g fractions
(DESM, 40; DSM, 47; paranodular tissues, 200 nmol H2O2/h/mg
protein). Thus, a decreased Ca2ⴙ/NAD(P)H-dependent H2O2
generation is the probable cause of the organification defect
in these goiters. (J Clin Endocrinol Metab 86: 4843– 4848, 2001)
I
In human thyroid tissues the presence of a Ca2⫹/
NAD(P)H-dependent H2O2 generator similar to the porcine
thyroid NADPH oxidase has only recently been characterized (13), and the thyroid oxidase (ThOx) cDNA has been
cloned (10, 11). However, further molecular identification of
the thyroid enzyme has not yet been achieved, and the
cDNAs cloned probably correspond only to the flavoprotein,
a component of the enzymatic system (14).
We report herein the study of two siblings with goiter and
hypothyroidism due to iodine organification defect. In these
goiters we found normal TPO activity and Tg content, but
thyroid NADPH oxidase activity was almost undetectable in
both the particulate 3,000 ⫻ g (P3,000 g) and 100,000 ⫻ g
(P100,000 g) fractions compared with the activity found in
paranodular to cold thyroid nodule tissues.
ODIDE IS RAPIDLY transported into thyroid follicular
cells and in the cellular apical surface is covalently bound
to Tg in an enzymatic reaction called iodine organification (1,
2). Failure of iodine organification results in impaired thyroid hormone biosynthesis, increased TSH stimulation, and
goiter (3, 4). In patients with goiter and hypothyroidism, a
thyroid iodine organification defect is suspected when intrathyroidal radiolabeled iodide is displaced by perchlorate
or thiocyanate administration, resulting in a positive perchlorate discharge test (3, 4).
Thyroid iodine organification depends on thyroid peroxidase (TPO) activity, which is modulated by the concentration of substrates (Tg and iodide) and cofactor (hydrogen
peroxide) (1). H2O2 generation is a limiting step in thyroid
hormone biosynthesis, as previously shown in dog thyroid
slice (5). In porcine thyroid glands previous biochemical
studies have reported that H2O2 is generated by a calciumdependent NAD(P)H:O2 oxidoreductase, the thyroid
NADPH oxidase (6 – 8). Some researchers have previously
shown that thyroid NADPH oxidase activity (9) and gene
expression (10, 11) are induced by TSH, as are TPO and Tg.
In dog thyrocytes, TSH also induces the expression of the
H2O2 generator via the cAMP cascade (12). NADPH oxidase,
therefore, seems to be the enzyme responsible for H2O2 generation linked to thyroid hormonogenesis.
Abbreviations: DTG, Diffuse toxic goiter; HRP, horseradish peroxidase; P3,000 g, particulate 3,000 ⫻ g; P100,000 g, particulate 100,000 ⫻
g; PAS, periodic acid-Schiff; PN, paranodular tissues; ThOx, thyroid
oxidase; TPO, thyroid peroxidase.
Materials and Methods
Materials
NADPH and lyophilized horseradish peroxidase (HRP; grade 1) were
purchased from Roche (Mannheim, Germany); BSA, scopoletin, cytochrome c, and FAD were obtained from Sigma (St. Louis, MO).
Patients
Family M, with no history of consanguinity, is composed of three
siblings; two of them are affected (Fig. 1), indicating that the patients can
be heterozygous for the mutation. The index patient (DSM), a 19 yr-old
male, underwent total thyroidectomy for mechanical neck compression
caused by the presence of a very large goiter. At 3 yr of age he started
thyroid hormone replacement due to thyroid enlargement associated
with clinical and laboratory findings of hypothyroidism. Hearing and
4843
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J Clin Endocrinol Metab, October 2001, 86(10):4843– 4848
mental development were normal. During adolescence his goiter increased in size. At the time of surgery a large multinodular asymmetrical
goiter with a fiber-elastic texture was noted. Thyroid autoantibodies
(anti-TPO and anti-Tg) were negative. Two-hour thyroid radioiodine
uptake was 63% (normal, 4 –12%), and potassium perchlorate (1.5 g,
orally) administration produced a discharge of 70% of the thyroid radioiodine content. Ultrasonographic studies of the thyroid confirmed
multinodular goiter, with several anechoic and hypoechoic areas. DSM
had been off thyroid medication for 30 d at the time of surgery.
His sister (DESM) had hypothyroidism diagnosed at 3 months of age,
when thyroid hormone replacement was started. She also had no mental
retardation or hearing loss, and did not maintain adequate adherence to
the prescribed medication. Goiter was first noted when she was 11 yr old,
and her thyroid enlarged considerably during adolescence. At admission to the university hospital at the age of 17 yr, a large soft multinodular asymmetrical goiter was noted. Thyroid autoantibodies (antiTPO and anti-Tg) were also negative. Thyroid radioiodine uptake (2 h)
was increased (77%; normal, 4 –12%), and she also had a positive perchlorate discharge test (50% of thyroid-accumulated radioiodine). Thyroidectomy was performed due to compressive symptoms, and the
patient was off thyroid hormone replacement for 30 d before surgery.
The patients gave their informed consent and the study was approved
by the institutional human research committee.
Serum total and free T4, T3, and TSH were assayed using commercial
kits (Euro/Diagnostic Products, Los Angels, CA). Anti-TPO and anti-Tg
autoantibodies and serum Tg were also assayed using commercial kits.
Laboratory data from both patients are described in Table 1. No cochlear
malformation was detected by computer axial tomography.
Thyroid tissue
Thyroid tissue samples from the dyshormonogenetic goiters were
obtained during thyroidectomy. Samples from paranodular tissues (PN)
surrounding cold thyroid nodules (n ⫽ 5) or a diffuse toxic goiter (DTG)
were used as controls. Analyses in the DTG sample were performed in
parallel with dyshormonogenetic goiters. Patients with cold thyroid
nodules received no medication before surgery, and the patient with
diffuse toxic goiter used propylthiouracil until the day before surgery
and saturated iodine solution (Lugol, five drops, three times per day)
14 d before surgery. When the DTG patient achieved euthyroidism,
thyroidectomy was performed. Thyroid tissue samples were either
freshly processed for H2O2 generation measurements or stored at ⫺20
C for other experiments.
Figueiredo et al. • Goiter due to Thyroid NADPH Oxidase Defect
For light microscopy, thyroid tissue samples were immediately fixed
in 10% buffered formalin. Sections of paraffin-embedded tissue were
stained with hematoxylin-eosin or periodic acid-Schiff (PAS).
Thyroid sample processing
For NADPH oxidase and cytochrome c reductase preparations, fresh
human thyroid tissue samples (1 g) were cleaned from fibrous tissue or
hemorrhagic areas, minced, and homogenized in sodium phosphate
buffer, pH 7.2, containing 0.25 m sucrose, 0.5 mm dithiothreitol, and
1 mm EGTA using an Ultra-Turrax (IKA, Staufen, Germany). The
homogenate was filtered through cheesecloth. The particulate fraction
was collected by centrifugation at 3,000 ⫻ g for 15 min at 4 C and
resuspended in 3 ml 50 mm sodium phosphate buffer, pH 7.2, containing
0.25 m sucrose and 2 mm MgCl2 (buffer A). The pellet was washed twice
with 3 ml buffer A and centrifuged at 3,000 ⫻ g for 15 min at 4 C. The
last pellet (P3,000 g) was gently resuspended in 1 ml buffer A. The
supernatant of the first centrifugation was centrifuged at 100,000 ⫻ g for
1 h at 4 C. The pellet (microsomal fraction, P100,000 g) was washed twice
in 2 ml buffer A and gently resuspended in 0.5 ml buffer A. Protein
concentrations were measured by the method of Bradford (15), using
BSA as standard. The particulate fractions (P3,000 g and P100,000 g) were
incubated with 1 n NaOH (30 min, 20 C) to dissolve particulate before
protein determination.
For TPO preparation, thyroid tissue samples (1 g) were cleaned from
fibrous tissue or hemorrhagic areas, minced, and homogenized in 50 mm
Tris-HCl buffer, pH 7.2, containing 1 mm potassium iodide, using an
Ultra-Turrax. The homogenate was centrifuged at 100,000 ⫻ g for 1 h at
4 C, and the pellet was resuspended in 2 ml digitonin (1%, wt/vol). The
mixture was incubated at 4 C for 24 h and then centrifuged at 100,000 ⫻
g for 1 h at 4 C. The supernatant containing solubilized TPO was used
for the iodide oxidation and albumin iodination assays, as previously
described (16 –18).
Ca2⫹- and NADPH-dependent H2O2 generating system:
NADPH oxidase activity
H2O2 formation was measured by incubating samples of the thyroid
particulate fractions (P3,000 g and P100,000 g) at 30 C in 1 ml 170 mm
sodium phosphate buffer, pH 7.4, containing 1 mm sodium azide, 1 mm
EGTA, 1 ␮m FAD, and 1.5 mm CaCl2, as previously described (8, 9, 13).
The reaction was started by adding 0.2 mm NADPH; aliquots of 100 ␮l
were collected at intervals up to 20 min and mixed with 10 ␮l 3 n HCl
to stop the reaction and destroy the remaining NADPH. Initial rates of
H2O2 formation were determined from eight aliquots of each assay by
following the decrease in 0.4 ␮m scopoletin fluorescence in the presence
of HRP (0.5 ␮g/ml) in 200 mm phosphate buffer, pH 7.8, in a spectrofluorometer (F 4000, Hitachi, Hialeah, FL), as previously described
(13). The excitation and emission wavelengths were 360 and 460 nm,
respectively. All measurements were performed on at least three samples from each particulate preparation and are expressed as nanomoles
of H2O2 per h. Specific activities were expressed per mg protein (nanomoles of H2O2 per h/mg protein) in the thyroid P3,000 g and P100,000
g fractions.
Thyroid NADPH-cytochrome c reductase activity
FIG. 1. Pedigree of the patient’s family. F, Female subjects studied;
f, male subjects studied; 䡺, normal male; E, normal female; L, female
with goiter.
Aliquots of human thyroid particulate fractions (P3,000 g and
P100,000 g) were incubated at 30 C in 1 ml 50 mm sodium phosphate
buffer (pH 7.2) containing 1 mm sodium azide, 1.2 mm EGTA, and 0.1
mm cytochrome c. The reaction was started by adding 0.1 mm NADPH.
The initial NADPH concentration was measured at 340 nm in a U-3300
(Hitachi) double beam spectrophotometer, using a molar absorption
TABLE 1. Laboratorial data from DESM and DSM patients
2-h
DESM
DSM
Normal range
131
I uptake
(%)
63
77
4 –12
Perchlorate test
(% of 131I discharged)
50
70
⬍15
Total T3
(ng/dl)
Total T4
(␮g/dl)
Free T4
(ng/dl)
TSH
(␮U/ml)
Serum Tg
(ng/ml)
64.4
72.6
1.2
1.8
0.23
0.22
694.0
12.5
⬎500
⬎500
80 –180
4.5–12.5
0.9 –1.9
0.47–5.0
⬍55
All data were obtained after suspension of T4 replacement for 30 days.
Figueiredo et al. • Goiter due to Thyroid NADPH Oxidase Defect
J Clin Endocrinol Metab, October 2001, 86(10):4843– 4848 4845
coefficient of 6.2 ⫻ 103 m/cm. Aliquots of 100 ␮l were taken at intervals
and mixed with 10 ␮l 2 mm dithiothreitol and 100 ␮l 2% SDS to stop the
reaction. The initial rates of cytochrome c-dependent NADPH oxidation
were determined from eight aliquots of each assay by following the
decrease in NADPH fluorescence at pH 8.0 in a Hitachi spectrofluorometer (F 4000), as previously described (9). The excitation and emission
wavelengths were 340 and 453 nm, respectively. NADPH-cytochrome c
reductase activity was expressed as nanomoles of NADPH oxidized per
h and milligrams of protein in the thyroid P3,000 g and P100,000 g
fractions (nanomoles of NADP⫹ per h/mg protein).
TPO iodide oxidation and albumin iodination activities
TPO iodide oxidation assays were performed using 12 mm potassium
iodide in 50 mm phosphate buffer (pH 7.4) and glucose-glucose oxidase
as the hydrogen peroxide (H2O2)-generating system, as previously described (16, 17). The increase in absorbency at 353 nm (⌬A353) was
followed for 4 min on a U-3300 Hitachi double beam spectrophotometer.
The TPO activity was estimated from the ⌬A353/min determined from
the linear portion of the reaction curve. One unit of iodide oxidation
activity is defined as ⌬A353/min, and activity was related to the protein
concentration in the enzyme preparation (units per g protein).
The TPO albumin iodination activity was determined using BSA as
iodine acceptor, and trichloroacetic acid precipitation of iodine bound
to BSA, as previously described (18). TPO iodination activity was expressed as nanomoles of I bound per mg protein/30 min.
Tg extraction and purification
Thyroid tissue samples (1 g) were homogenized and centrifuged at
100,000 ⫻ g at 4 C for 1 h. The supernatant containing soluble proteins
were submitted to salting out with 50% ammonium sulfate (4 m, pH 7.2),
to precipitate Tg. After a 24-h incubation period samples were centrifuged (10,000 ⫻ g, 4 C, 30 min), and the pellets were resuspended in 1
ml 50 mm sodium phosphate buffer, pH 7.4. Purified Tg was submitted
to dialysis (4 C, overnight), followed by protein measurement by the
method of Bradford (15). Tg aliquots were either used for TPO iodination
assays or SDS-PAGE gel electrophoresis (6.0% polyacrylamide), using
the method of Laemmli (19). Equal amounts of Tg protein (25 ␮g) were
applied and then stained by Coomassie blue.
Results
TPO iodide oxidation and albumin iodination activities in
the goiters of hypothyroid patients were higher than the
normal range found in previous reports from our laboratory
(16, 17) and did not differ significantly from the TPO activity
found in the DTG sample studied in parallel (Table 2).
Normal amounts of Tg could be extracted from the
goitrous tissues (DESM, 28; DSM, 17; DTG, 19 mg Tg/g
tissue), and their purified Tg could be normally iodinated in
vitro (DESM, 3.4; DSM, 4.3; DTG, 6.3 nmol I/mg Tg). Also,
SDS-PAGE analysis showed that Tg extracted from these
goiters was of normal mol wt.
The routine histological staining showed a similar general
appearance of thyroid architecture in both patients (Fig. 2).
Single layers of epithelial cells form spherical structures, the
TABLE 2. Thyroperoxidase activity found in DESM and DSM
goiters compared with TPO obtained from diffuse toxic goiters
(DTG) and paranodular tissue (PN)
TPO activity
DESM
DSM
PN
DTG
Iodide oxidation
(U/g protein)
Albumin iodination
(nmol I/mg protein)
1034
1064
232 ⫾ 121
2267
16
8
24a
66
a
Mean of two normal tissues previously published by our group in
Ref. 16.
thyroid follicles, and delimit the follicle lumens, which contain Tg. Although the follicles exhibit variable size and shape,
there is a predominance of hyperactive follicles, with high
epithelial cells and variable colloid quantity, especially in the
tissue sample from DESM.
In PN thyroid tissues, the NAPDH oxidase H2O2-generating levels found in the P3,000 g fraction (224 ⫾ 38 nmol
H2O2/h/mg protein) were similar to those described for
porcine thyroid particulate (6, 8). On the other hand, H2O2
generation was either undetectable (DESM) or very low
(DSM) in the P3,000 g (Fig. 3A) and low in the P100,000 g (Fig.
3B) obtained from DESM and DSM.
Cytochrome c reductase is another enzymatic system that
was proposed as capable of generating H2O2 in the thyroid
gland. We found high cytochrome c reductase activity compared with PN tissues in either the P3,000 g or P100,000 g
fraction (Fig. 4, A and B).
Discussion
In family M, the early onset of hypothyroidism together
with goiter development and a positive perchlorate discharge test suggest an inherited defect in thyroid iodine
organification, as previously reported for other families (20 –
22). The mode of inheritance remains unclear. By history,
there is no suggestion of a common ancestor, and this is
supported by genotyping several loci using microsatellite
markers (Carvalho, D. P., and P. Kopp, unpublished results).
A recessive mode of inheritance with homozygosity or compound heterozygosity for a defective gene cannot be excluded. Alternatively, the two affected siblings could carry
only one defective allele, exerting a dominant negative effect
or resulting in haploinsufficiency. Lastly, although less
likely, one should also consider the possibility that inactivation of one allele in two (or several) distinct genes could
be involved in the pathogenesis of the disorder.
Usually, Tg defects are described as the cause of dyshormonogenesis with a negative perchlorate discharge test. In
our patients a Tg defect can be excluded, because, apart from
the finding of positive perchlorate discharge, their Tg was
normally produced and could be normally iodinated by TPO
in vitro. Furthermore, the histological analysis shows normal
follicle structures with Tg detected by PAS in the lumen, also
excluding the possibility of a Tg transport defect as previously reported (23–25).
In Pendred’s syndrome it is believed that the cause of the
iodine organification defect is impaired iodide transport
through pendrin, a chloride/iodide transporter found in the
apical thyroid plasma membrane (26, 27). However, in Pendred’s syndrome perchlorate is usually only partially discharged, the patients have congenital sensorineural deafness,
and most of them are euthyroid (28). In contrast, our patients
are hypothyroid, have a complete organification defect, as an
almost complete perchlorate discharge (66% and 77%) was
detected, and have no signs of cochlear malformation, indicating that pendrin might be normal in these two goitrous
patients.
Defective TPO is believed to be the most common cause of
a positive perchlorate discharge test in thyroid dyshormonogenesis (22). However, in some patients with iodine or-
4846
J Clin Endocrinol Metab, October 2001, 86(10):4843– 4848
Figueiredo et al. • Goiter due to Thyroid NADPH Oxidase Defect
FIG. 2. Light micrographs of thyroid
tissue. Thyroid tissue samples from the
goiters of two hypothyroid siblings,
DSM (B and C) and DESM (A and D),
were fixed in 10% buffered formalin.
Sections of paraffin-embedded tissue
were stained with hematoxylin eosin
(A) or PAS (B–C). A and B, Typical thyroid follicular structure and stroma (S)
with irregular collagenous connective
tissue. Follicular size and shape are
heterogeneous. The colloid (C) is PAS
positive. C and D, Some hyperactive follicles with hyperplasia of the follicular
epithelial cells (FC) and regions of colloid endocytosis (*). The basal lamina
(BL) that unsheathes each follicle is
also PAS positive. A, ⫻100; B, ⫻40; C
and D, ⫻400.
ganification defects and normal TPO, impaired H2O2 generation has been proposed (16, 29, 30).
Kusakabe (30) has described a defect in thyroid cytochrome b5 reductase as the cause of a goiter with H2O2
generation defect, but the participation of this enzyme in
thyroid H2O2 generation has not been confirmed. The nature
of the enzymatic system involved in thyroid H2O2 production has only recently been determined (10, 11, 13), and
current data strongly indicate that H2O2 linked to thyroid
hormonogenesis is generated by Ca2⫹- and NAD(P)H-
dependent ThOx (NADPH oxidase or ThOx). In the present
study we found normal TPO and decreased NADPH oxidase
activity in both siblings, reinforcing the possibility that their
goiters might be caused by a defect in H2O2 generation.
Recently, two cDNAs (ThOx 1 and ThOx 2) that encode ThOx
have been cloned (10, 11), but they correspond to the flavoprotein, a component of the H2O2-generating enzymatic
system that is responsible for NADPH oxidation, but not for
the electron transfer necessary for H2O2 production (14).
Hence, as biochemical data suggest that thyroid H2O2 is
Figueiredo et al. • Goiter due to Thyroid NADPH Oxidase Defect
J Clin Endocrinol Metab, October 2001, 86(10):4843– 4848 4847
NAD(P)H:O2 oxidoreductase is involved in H2O2 generation
associated with TPO and participates in the iodine organification reaction.
Acknowledgments
Received March 22, 2001. Accepted June 26, 2001.
Address all correspondence and requests for reprints to: Dr. Denise
Pires de Carvalho, Instituto de Biofı́sica Carlos Chagas Filho, CCS-Bloco
G, Cidade Universitária, Ilha do Fundão, 21949-900 Rio de Janeiro,
Brazil. E-mail: [email protected].
FIG. 3. Thyroid NADPH oxidase activity. Ca2⫹- and NADPH-dependent H2O2 generation was measured in human thyroid samples obtained from five paranodular to cold nodule tissues (PN) and from two
siblings with goiter and hypothyroidism (DESM and DSM). H2O2
generation was measured in 170 mM sodium phosphate buffer, pH 7.4,
containing 1 mM sodium azide, 1 mM EGTA, 1 ␮M FAD, and 1.5 mM
CaCl2. The reaction was started by adding 0.2 mM NADPH; aliquots
of 100 ␮l were collected at intervals up to 20 min and mixed with 10
␮l 3 N HCl to stop the reaction. Initial rates of H2O2 formation were
determined from eight aliquots of each assay by following the decrease
in 0.4 ␮M scopoletin fluorescence in the presence of HRP (0.5 ␮g/ml)
in 200 mM phosphate buffer, pH 7.8, in a Hitachi spectrofluorometer
(F 4000; excitation, 360 nm; emission, 460 nm). A, P3,000 g fraction;
B, microsomal fraction (P100,000 g). Enzyme activity is expressed as
mean of at least two measurements in each particulate preparation.
FIG. 4. Thyroid cytochrome c reductase. NADPH oxidation by cytochrome c reductase and TPO iodide oxidation activities were measured in human thyroid samples obtained from five paranodular to
cold nodule tissues (PN) and from two siblings with goiter and hypothyroidism (DESM and DSM). Cytochrome c reductase activity was
measured in the presence of 1 mM sodium azide, 1.2 mM EGTA, and
0.1 mM cytochrome c. The reaction was started by adding 0.1 mM
NADPH; aliquots of 100 ␮l were taken at intervals and mixed with
10 ␮l 2 mM dithiothreitol and 100 ␮l 2% SDS to stop the reaction.
NADPH oxidation was determined from eight aliquots of each assay
by following the decrease in NADPH fluorescence at pH 8.0 in a
Hitachi spectrofluorometer (F 4000; excitation, 340 nm; emission, 453
nm). A, P3,000 g; B, microsomal fraction (P100,000 g). All measurements were performed on at least two samples from each particulate
preparation.
generated by a multicomponent enzyme, it is possible that
other components of the enzymatic system responsible for
H2O2 production could be involved in the impaired hormonogenesis in these goiters. Moreno et al. (31) recently described possible inactivating mutations of the ThOx2 gene in
some cases of congenital hypothyroidism; however, functional studies to determine the implications of these mutations on partial iodine organification defects are still lacking.
In conclusion, this is the first report of a family with goiter
and hypothyroidism due to impaired NADPH oxidase activity. We have shown that the thyroid calcium-dependent
References
1. Taurog A 1991 Hormone synthesis: thyroid iodine metabolism. In: Braverman
LE, Utiger RD, ed. The thyroid, 6th Ed. New York: Lippincott; 51–97
2. DeGroot LJ, Niepomniszcze H 1977 Biosynthesis of thyroid hormone: basic
and clinical aspects. Metabolism 26:665–718
3. Refetoff S, Dumont J, Vassart G 2001 Thyroid disorders. In: Scriver CR,
Beaudet AL, Sly WS, eds. The metabolic and molecular bases of inherited
disease, 8th Ed, International Edition. New York: McGraw-Hill; 4029 – 4075
4. Medeiros-Neto G, Stanbury JB 1994 Defective organification of iodide. In:
Medeiros-Neto G, Stanbury JB, eds. Inherited disorders of the thyroid system.
Boca Raton: CRC Press; 53– 80
5. Corvilain B, Van Sande J, Laurent E, Dumont JE 1991 The H2O2 generating
system modulates protein iodination and the activity of the pentose phosphate
pathway in dog thyroid. Endocrinology 128:779 –785
6. Virion A, Michot JL, Dëme D, Kaniewski J, Pommier J 1984 NADPHdependent H2O2 generation and peroxidase activity in thyroid particulate
fraction. Mol Cell Endocrinol 36:95–105
7. Dupuy C, Kaniewski J, Dème D, Pommier J, Virion A 1989 NADPH-dependent H2O2 generation catalyzed by thyroid plasma membranes. Studies with
electron scavengers. Eur J Biochem 185:597– 603
8. Dème D, Virion A, Aôt-Hammou N, Pommier J 1985 NADPH-dependent
generation of H2O2 in a thyroid particulate fraction requires Ca⫹2. FEBS Lett
186:107–110
9. Carvalho DP, Dupuy C, Gorin Y, et al. 1996 The Ca⫹2- and reduced nicotinamide adenine dinucleotide phosphate-dependent hydrogen peroxide generating system is induced by thyrotropin in porcine thyroid cells. Endocrinology 137:1007–1012
10. Dupuy C, Ohayon R, Valent A, Noel-Hudson MS, Dème D, Virion A 1999
Purification of a novel flavoprotein involved in the thyroid NADPH oxidase.
Cloning of the porcine and human cDNAs. J Biol Chem 274:37265–37269
11. De Deken X, Wang D, Many MC, et al. 2000 Cloning of two human thyroid
cDNAs encoding new members of the NADPH oxidase family. J Biol Chem
275:23227–23233
12. Raspé E, Dumont JE 1995 Tonic modulation of dog thyrocyte H2O2 generation
and I⫺ uptake by thyrotropin through the cyclic adenosine 3⬘,5⬘-monophosphate cascade. Endocrinology 136:965–973
13. Leseney AM, Dème D, Dupuy C, et al. 1999 Biochemical characterization of
a Ca2⫹/NAD(P)H-dependent H2O2 generator in human thyroid tissue. Biochimie 81:373–380
14. Gorin Y, Ohayon R, Carvalho DP, et al. 1996 Solubilization and characterization of a thyroid Ca2⫹-dependent and NADPH-dependent K3Fe(CN)6 reductase. Relationship with the NADPH-dependent H2O2 generating system.
Eur J Biochem 240:807– 814
15. Bradford MM 1976 A rapid and sensitive method for he quantification of
microgram quantities of proteins utilizing the protein-dye binding. Anal Biochem 72:248 –252
16. Carvalho DP, Rego KGM, Rosenthal D 1994 Thyroid peroxidase in dyshormonogenetic goiters with organification and thyroglobulin defects. Thyroid
4:421– 426
17. Moura EG, Rosenthal D and Carvalho-Guimarães DP 1989 Thyroid peroxidase activity in human nodular goiters. Brazilian J Med Biol Res 22:31–39
18. Carvalho-Guimarães DP, Ramos CF, Rosenthal D 1989 A technical improvement for the thyroid peroxidase iodination assay. Brazilian J Med Biol Res
22:821– 823
19. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227:680 – 685
20. Lever EC, Medeiros-Neto GA, DeGroot L 1983 Inherited disorders of thyroid
metabolism. Endocr Rev 4:213–239
21. Takeuchi K, Suzuki H, Yoshihiko H, Mashimo K 1970 Significance of iodideperchlorate discharge test for detection of iodine organification defect of the
thyroid. J Clin Endocrinol Metab 31:144 –146
22. Medeiros-Neto GA, Billerbeck AEC, Wajchenberg BL, Targovnik HM 1993
Defective organification of iodide causing hereditary goitrous hypothyroidism. Thyroid 3:143–159
4848
J Clin Endocrinol Metab, October 2001, 86(10):4843– 4848
23. Ohyama Y, Hosoya T, Kameya T, et al. 1994 Congenital euthyroid goitre with
impaired thyroglobulin transport. Clin Endocrinol (Oxf) 41:129 –135
24. Kim PS, Kwon OY, Arvan P 1996 An endoplasmic reticulum storage disease
causing goiter with hypothyroidism. J Cell Biol 133:517–527
25. Medeiros-Neto G, Kim PS, Yoo SE, et al. 1996 Congenital hypothyroid
goiter with deficient thyroglobulin. Identification of an endoplasmic reticulum storage disease with induction of molecular chaperones. J Clin Invest
98:2838 –2844
26. Bidart JM, Mian C, Lazar V, et al. 2000 Expression of pendrin and the Pendred
syndrome (PDS) gene in human thyroid tissues. J Clin Endocrinol Metab
85:2028 –2033
27. Royaux IE, Suzuki K, Mori A, et al. 2000 Pendrin, the protein encoded by the
Figueiredo et al. • Goiter due to Thyroid NADPH Oxidase Defect
28.
29.
30.
31.
Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and
is regulated by thyroglobulin in FRTL-5 cells. Endocrinology 141:839 – 845
Kopp P 1999 Pendred’s syndrome: identification of the genetic defect a century
after its recognition. Thyroid 9:65– 69
Niepomniszcze H, Targovnik HM, Gluzman BE, Curutchet P 1987 Abnormal
H2O2 supply in the thyroid of a patient with goiter and iodine organification
defect. J Clin Endocrinol Metab 65:344 –348
Kusakabe T 1975 Deficient cytochrome b5 reductase activity in nontoxic goiter
with iodide organification defect. Metabolism 24:1103–1113
Moreno JC, Bikker H, de Randamie J, et al. 2000 Mutations in the ThOx2 gene
in patients with congenital hypothyroidism due to iodide organification defects. Endocr J 47:107
Project Announcement
Genetically Modified Animals in Endocrinology
As a service to the endocrine community, Endocrine Reviews intends to publish bibliographies of papers
describing knockout, transgenic, and mutant animals that may be useful in the study of endocrinology. In
the print version of the journal, we will publish subject-limited bibliographies as the individual sections
become available. We also intend to create a cumulative database to be made available on the web in a
searchable format. At this time, we would like to hear what enhancements would be desirable on this web
site.
Readers are encouraged to contact the editorial office with bibliographic information about knockout,
transgenic, and mutant animals that they would wish to have included in the database; please include the
species and the citation for the article in which the original description appeared. In addition, suggestions
regarding topics that we should consider adding to our bibliographies would be appreciated.
Address your contributions to the database via e-mail or standard mail, using the following addresses:
Dr. E. Brad Thompson/Endocrine Reviews, The University of Texas Medical Branch, Room 111C, Basic
Science Building, Galveston, TX 77555-0628 USA. [email protected]