European Journal of Endocrinology (2003) 149 247–256
`
`ISSN 0804-4643
`
`EUROPEAN THYROID ASSOCIATION HENNING PRIZE LECTURE, Goteborg, 10 September 2002
`
`Primary congenital hypothyroidism: defects in iodine pathways
`Jan J M de Vijlder
`Department of Paediatric Endocrinology, Emma Children’s Hospital AMC, Academic Medical Centre, University of Amsterdam, Amsterdam,
`The Netherlands
`
`(Correspondence should be addressed to Jan J M de Vijlder; j.j.devijlder@amc.uva.nl)
`
`Abstract
`
`The thyroid gland is the only source of thyroid hormone production. Thyroid hormone is essential for
`growth and development, and is of special importance for the development of the central nervous
`system. It was for that reason that neonatal screening on congenital hypothyroidism was introduced
`and is now performed in many countries. Defects in thyroid hormone production are caused by sev-
`eral disorders in hormone synthesis and in the development of the thyroid gland (primary hypothy-
`roidism) or of the pituitary gland and hypothalamus (central hypothyroidism).
`This paper describes defects in the synthesis of thyroid hormone caused by disorders in the synthesis
`or iodination of thyroglobulin, leakage of iodinated proteins by a stimulated thyroid gland and the
`presence of abnormal iodoproteins, mainly iodinated albumin, in the thyroid gland and blood circula-
`tion. Circulating thyroglobulin and abnormal iodoproteins, as well as the breakdown products of
`these iodoproteins excreted in urine, are used for etiological diagnosis and classification. Moreover,
`our finding of an enzyme that catalyses the dehalogenation of iodotyrosines, which is important
`for iodine recycling and required for economical use of iodine, is also referred to.
`
`European Journal of Endocrinology 149 247–256
`
`Introduction
`
`Thyroid hormone, exclusively produced by the thyroid
`gland, plays a key role in the growth and differentiation
`of many organs. It is of special importance for the devel-
`opment of the central nervous system in the pre- and
`postnatal period (1, 2). For thyroid development and
`for synthesis, storage and secretion of thyroid hormone
`a variety of transcription factors and a sequence of pre-
`cisely tuned events (3) are required. Disorders in one of
`these factors or in the intermediate reaction steps are
`the molecular basis of abnormalities in thyroid develop-
`ment and/or in thyroid hormonogenesis that result in
`congenital hypothyroidism. Severe shortage of thyroid
`hormone after birth from several weeks on will result
`in serious mental and motor handicaps. The initial
`reports on the absence of thyroid tissue leading to
`sporadic cretinism and myxoedema, and the occur-
`rence of endemic cretinism due to iodine deficiency
`are from the middle of the 19th century. The first
`descriptions of
`inborn errors in thyroid metabolism
`causing hypothyroidism with goitre are from Pendred
`(4) and Osler (5) at the end of the 19th century. Never-
`theless, even in the middle of the twentieth century,
`many patients with congenital hypothyroidism were
`referred to paediatricians at school age (6).
`Apart from the children with the classical type of
`congenital hypothyroidism,
`there are also special
`
`groups of children who are in danger from a shortage
`of thyroid hormone after birth such as preterm born
`infants (7) and infants with Down’s syndrome (8).
`During pregnancy the mother provides substantial
`amounts of thyroid hormone to the foetus (9), so the
`delay in cerebral development caused by congenital
`hypothyroidism (CH) is mainly the result of postnatal
`thyroid hormone deficiency. The
`result of
`the
`maternal thyroid hormone provision to the foetus is
`that hypothyroid newborns do not show clear clinical
`signs at birth. The risk of mental retardation and the
`difficulty in recognising the disease were reasons for
`introducing neonatal mass screening programmes in
`many countries. The introduction was attended by a
`considerable increase in the apparent overall incidence
`of CH, most likely due to the recognition of mild dis-
`orders
`that
`formerly remained undetected, were
`detected later and/or were not recognised as congenital
`problems (10, 11).
`Congenital hypothyroidism can be of thyroidal or of
`central
`(hypothalamic/pituitary) origin (Table 1),
`with a widely diverse molecular aetiology. Almost all
`screening programmes are directed towards the detec-
`tion of thyroidal congenital hypothyroidism (incidence
`about 1:3100) (12) by measuring thyrotrophin (TSH)
`in filter blood spots, and they ignore
`central
`congenital hypothyroidism, which has an estimated
`incidence of about 1:20 000 births (12 –14). In this
`
`q 2003 Society of the European Journal of Endocrinology
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`J J M de Vijlder
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`Table 1 Congenital hypothyroidism.
`
`Thyroidal congenital hypothyroidism
`Disorders in development of the thyroid gland
`- thyroid is absent
`- under-development with migration failure
`- under-development with normal migration
`Disorders in thyroid hormone synthesis
`- TSH hypo-responsiveness.
`- defects in iodide transport from circulation into the thyroid
`cell
`- defects in iodide transport from the thyroid cell to the
`follicular lumen, often combined with inner ear deafness
`(Pendred syndrome)
`- defects in the synthesis of hydrogen peroxide
`- defects in the oxidation of iodide, iodination and
`iodothyronine synthesis
`- defects in processes involved in the synthesis or
`degradation of thyroglobulin
`- detects in iodine recycling
`Central congenital hypothyroidism
`Disorders in development and/or function of the
`- hypothalamus
`- pituitary gland
`- both
`
`paper, I mainly describe disorders in the mechanism of
`thyroid hormone synthesis taking place at the apical
`membrane of the thyroid gland.
`
`Thyroid metabolism
`
`Iodination takes place at the apical membrane (see
`oval
`in Fig. 1). The active transport of
`iodide into
`þ
`-I2 sympor-
`the follicular cells is mediated by the Na
`ter (NIS) as the first crucial step in thyroid hormone
`
`EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) 149
`
`synthesis (for review see (15)). Iodide taken up by
`the thyrocytes is transported through the apical
`membrane by the anion transporter, pendrin (16).
`Recently, another apical
`iodide transporter protein
`(AIT) (17) has been described, which was identified
`as an NIS homologue.
`The oxidation of iodide requires hydrogen peroxide
`that
`is synthesised outside the cell at
`the apical
`border catalysed by the thyroid – oxidase complex.
`Recently, two proteins of this complex ThOX1 and
`ThOX2 have been identified (18). The iodination of
`tyrosine residues, commonly referred to as iodide
`organification,
`is catalysed by the membrane-bound
`thyroperoxidase (TPO). Besides the binding of iodine
`to tyrosine, TPO also catalyses the coupling of iodotyr-
`osines to iodothyronine residues in thyroglobulin (TG)
`under oxidative conditions. In order to deliver thyroid
`hormone to the blood circulation, TG is internalised
`by endocytosis that can take place by non-selective
`fluid phase uptake and by receptor-mediated processes.
`Two receptors playing a role in the endocytosis of TG
`have been identified: megalin (19) and the thyroid asia-
`loglycoprotein (20) receptor. After endocytosis, thyrox-
`ine (T4),
`triiodothyronine (T3) and the remaining
`iodotyrosine residues in TG are released by lysosomal
`proteolysis. The iodothyronines are secreted into the cir-
`culation, non-covalently bound to plasma proteins and
`transported to target organs. The iodotyrosine molecules
`are deiodinated and the iodide can be reused as substrate
`for the iodinating processes in the thyroid.
`In all these steps leading to the synthesis and release
`of thyroid hormone and the recycling of iodine, defects
`may occur (Table 2).
`
`Figure 1 (A) Section of a normal thyroid gland stained with haematoxylin-eosin and immuno-stained with anti-thyroglobulin.
`(B) Schematic drawing of a thyroid cell with the main iodination pathways. Modified from Van de Graaf et al. (24). IGF-1, insulin-like
`growth factor I; IGF-1-R, IGF-I receptor; TSH-R, thyrotrophin receptor; N, nucleus; M, mitochondria; L, lysosomes; E, endosomes;
`TJ, tight junction.
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`Defects in iodine pathways 249
`
`Table 2 Disorders in thyroid hormone synthesis.
`
`Class of defect
`
`Features
`
`Thyroglobulin
`synthesis
`defects
`
`Presence of iodoalbumin in plasma and/or
`iodopeptides in urine. Plasma TG* conc. Not
`related to TSH plasma conc.
`
`Iodide
`transport**
`
`Through basal-lateral membrane. Saliva/blood
`ratio of iodide near unity
`Through apical membrane*** deafness
`
`Iodide
`organification
`defect**
`
`Partial** (PIOD) deafness
`
`Partial defect (PIOD)
`
`Total (TIOD)
`
`Plasma TSH
`conc.
`
`Plasma T4
`conc.
`
`Elevated
`
`Low-normal
`
`123I2 uptake 1 wash-out****
`
`Putative mol. defects
`
`Elevated uptake. Partial wash-out possible.
`Rapid turnover dependent on amount of TG
`in follicular lumen
`
`A: Defects in TG-synthesis pathway
`B: Mutations in TG gene
`
`Elevated
`
`Low
`
`No uptake
`
`Normal or
`elevated
`
`Normal-
`elevated
`Normal-
`elevated
`Elevated
`
`Low or normal Partial wash-out
`
`Low-normal
`
`Partial wash-out
`
`Low-normal
`
`Partial wash-out
`
`Low
`
`Total wash-out
`
`Sodium-iodide symporter (NIS)
`Pendrin (PDS gene) þ apical iodide
`transporter (AIT) and/or possibly
`other transporters?
`
`Thyroid oxidase (ThOX2),
`monoallelic mutation
`ThOX2 or TPO biallelic mutations
`
`Iodine recycling
`defect
`
`Presence of mono- and di-iodotyrosines in urine Variable,
`dependent on
`iodine status
`
`Variable,
`dependent on
`iodine status
`
`High uptake, with rapid turnover
`
`DEHALI
`
`Conc., concentration; PIOD, partial organification defect; TIOD, total organification defect.
`* In thyroid by dyshormonogenesis elevated plasma TSH concentrations, stimulating the thyroid gland, result in elevated plasma TG concentrations, except in the cases of TG synthesis defects and TSH
`receptor inactivation where elevated TSH concentrations do not give increased plasma TG concentrations.
`** Pendred’s syndrome is characterised by a combined thyroid defect and deafness.
`*** Pendred’s syndrome is probably caused by an iodide transport defect over the apical membrane, radio-iodine uptake studies reflect a partial organification defect.
`**** 123I taken up that remains as iodide in the thyroid gland will be released rapidly when the NIS transporter is blocked by e.g. perchlorate ions. When the release is between 10% and 90% or . 90% in half
`an hour it is called a partial or total wash-out respectively.
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`Disorders in thyroid hormone synthesis
`
`Thyroglobulin synthesis
`
`In the process of thyroid hormone synthesis and sto-
`rage, TG plays a prominent role. TG is encoded by a
`large gene, mapped to human chromosome 8q24,
`spanning more than 300 kb (21) containing 48 exons
`(22). The primary structure of human TG was deduced
`from the sequence of
`its 8448-base mRNA (23).
`The open reading frame for human TG consists of
`8307 nucleotides coding for a protein of 2768 amino
`acid residues which has 66 tyrosine residues and a
`signal peptide of 19 amino acids (24). The TG sequence
`contains several repeats – in the N-terminal half eleven
`type-1 cysteine repeats were described (25). These
`repeats have been reported in other proteins and may
`play a role in the self-folding of proteins or may act
`as a proteinase inhibitor (26).
`The structure of TG is important for optimal T4 pro-
`duction. The protein-dimer (molecular mass 660 000)
`consists of two identical monomers containing 10%
`carbohydrates. Four to five tyrosine residues (acceptor
`residues) in the TG monomer, which can be preferen-
`tially iodinated (27), are considered to couple with
`other specific iodo-tyrosine residues (donor residues)
`to form iodothyronine. The sites in the TG molecule
`where iodothyronine residues are formed are the so-
`called hormonogenic sites. An important hormonogenic
`site is the N-terminal tyrosine residue (tyr 5; acceptor)
`that most likely couples with iodotyrosine 130 as
`donor.
`It can be expected that mutations causing
`small changes in structure already influence coupling
`efficiency and, as a consequence, thyroid hormone syn-
`thesis and release (28). Even amino acid changes, con-
`sidered to be polymorphic, may influence the efficiency
`of
`thyroid hormone synthesis, changes that may
`become important under iodine deficient conditions.
`
`Thyroglobulin synthesis defects
`
`The characterization of TG synthesis defects is not easy
`to establish. The physiological characteristics leading to
`this classification are shown in Table 2. The concen-
`trations of TSH and thyroid hormones as well as the
`iodine uptake studies are indicative but one of the para-
`meters important for the diagnosis is the relatively low
`plasma TG concentration in relation to the plasma TSH
`level. In practice, the definition ‘TG synthesis defect’ is a
`clinical-physiological one. It does not cover defects in
`the gene coding for TG, but includes defects in the
`whole process of TG synthesis (29).
`
`Thyroglobulin synthesis defects in animals Before
`studies with recombinant DNA and transgenic mice
`became possible, the availability of animals with inheri-
`ted congenital hypothyroidism and goitre proved very
`
`www.eje.org
`
`useful as models for the study of disorders in TG
`synthesis.
`Five animals with TG synthesis defects have been
`described, and four are elucidated at the molecular
`level. The Afrikander cattle (30) in which affected ani-
`mals were borderline euthyroid required special care for
`normal
`growth,
`development
`and
`reproduction.
`The molecular defect appeared to be caused by a non-
`sense mutation (Arg697Stop) in exon 9 in the gene
`coding for TG. This mutation gives rise to a thyroglobu-
`lin fragment with a molecular mass of 75 000 plus an
`alternatively spliced TG monomer (molecular mass
`250 000), missing exon 9 (31, 32).
`The Australian Merino sheep (33) were severely
`hypothyroid and most lambs were either stillborn or
`died soon after birth. In the thyroid gland and blood cir-
`culation, abnormal iodinated proteins were observed,
`among which was iodinated albumin. In the thyroid
`gland no TG (molecular mass: 660 000) could be
`found. The defect inherited in an autosomal recessive
`way (34).
`In the cog/cog mouse, congenital goitre is linked to
`the TG locus and is caused by a Leu2366Pro transition
`in the acetylcholesterase domain of the TG molecule
`(35). This mutation resulted in an endoplasmic reticu-
`lum (ER) storage disease.
`Another mutation in the TG molecule that inherited
`in an autosomal recessive way has been found in
`severely hypothyroid Rdw rats (plasma T4 concen-
`tration: 15 nmol/l; normal: 64 nmol/l, TSH concen-
`tration: 9.8 ng/ml; normal: 1.1 ng/l). DNA sequencing
`revealed a Gly2320Arg mutation in the acetylcholin-
`esterase-like
`domain. The molecular
`chaperones
`GRP94, GRP78 and calreticulin were accumulated in
`the thyroid gland of these animals. TG was not secreted
`into the follicular lumina, but was retained in the
`dilated ER (36).
`The Dutch goats, a mixed strain of Saanen and dwarf
`goats, were severely hypothyroid. Almost all affected
`goats required thyroid hormone treatment or extra
`iodide administration to survive (37). The defect is
`inherited in an autosomal recessive way (38). The
`molecular defect in the TG gene was localised in exon
`8 caused by a TAC(Tyr)/296/TAG(Stop) mutation
`(39). The mutation gives rise to a cys-rich N-terminal
`TG peptide fragment with a molecular mass of
`35 kDa, and about 40 kDa when glycosylated, as in
`the in vivo situation. These TG fragments are trans-
`ported to the follicular lumen, conjugated by disulphide
`bridges to high molecular weight complexes from
`which the tyrosine residues can be iodinated and
`coupled to thyroid hormone residues when more than
`the normal amount of iodine was administered, render-
`ing the animals euthyroid (40, 41). For the neonates, it
`was very beneficial to give the mothers iodide (37, 42).
`
`Five
`Thyroglobulin synthesis defects in man
`mutations in the TG gene have been described in
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`Defects in iodine pathways 251
`
`human pathology. A homozygous C886T transition in
`exon 7 results in the replacement of an Arg residue
`into a premature stop codon at amino acid position
`277 in TG upon translation (43). Clinically, hypothy-
`roidism, observed in three related patients, was not
`severe indicating that the short TG fragment was still
`able to produce thyroid hormone, a phenomenon that
`was also observed in the goat model and in in vitro
`experiments
`(44).
`In these
`experiments
`it was
`shown that
`the preferential coupling of
`iodinated
`tyrosine residues 5 and 130 was still present, but less
`pronounced than in intact TG.
`In a Japanese family a deletion of 68 amino acids
`from the N-terminal part of thyroglobulin, due to an
`in-frame mutation of exon 4 was observed (45). The
`resulting TG molecule lacks the donor tyrosine residue
`130 and a cys-containing repeated sequence character-
`istic for the first half of the molecule. Consequently, this
`patient was severely hypothyroid.
`In a Brazilian family, two hypothyroid patients with
`large goitres were described. Investigation of these goi-
`tres revealed that in the TG gene a CGA(Arg)1510
`TGA(stop) transition gave rise to a truncated TG poly-
`peptide of 166 260 Da. Besides this mutation, an
`alternative splice product was present (46) that is also
`found in normal thyroid tissue, resulting in a TG lack-
`ing 57 amino acids.
`Another family has been described in which the TG
`mRNA lacked 138 nt between position 5590 and
`5727. The severely hypothyroid patients, homozygous
`for
`this deletion, presented multinodular goitres.
`Despite high TSH levels the serum TG level was below
`the detection level (, 1.0 mg/l) and no increase could
`be observed after TSH stimulation (47). The disease in
`the affected patients was associated with massive
`induction of specific ER molecular chaperones includ-
`ing the hsp90 homologue, GRP94, and the hsp70
`homologue, BiP. From thyroid tissues of
`the four
`patients, light microscopy demonstrated the presence
`of intracellular TG despite its absence in thyroid follicle
`lumina, while electron microscopy indicated abnormal
`distension of
`the endoplasmic reticulum. The data
`suggest that these patients synthesise a mutant thyro-
`globulin which is defective for folding/assembly, leading
`to a markedly reduced ability to export the protein from
`the ER to the follicular lumen (48). Thus, these kin-
`dreds suffer from a thyroid ER storage disease, a cell
`biological defect phenotypically indistinguishable from
`that found in cog/cog mice (35).
`Patients containing a Cys1263Arg mutation (28) are
`described as having an arrest in the endoplasmic reticu-
`lum as well. These patients were mildly hypothyroid to
`euthyroid, indicating that in the follicular lumen suffi-
`cient TG had to be iodinated to deliver adequate
`amounts of thyroid hormone.
`In Fig. 2, the locations of the various mutations in
`the TG protein molecule in man and animals are
`shown schematically.
`
`From the localisation of the mutations, we get the
`impression that patients producing shorter TG frag-
`ments are less severely hypothyroid than the patients
`having mutations in the acetylcholesterase domain of
`TG, indicating that shorter TG fragments are secreted
`more easily into the follicular lumen than TG molecules
`with more C-terminal mutations.
`The patient where exon 4 was spliced out lacks a Cys-
`containing repeat and Tyr residue 130. This patient was
`also severely hypothyroid. Thyroid dyshormonogenesis
`might be caused because the Tyr130 donor residue
`was missing. Otherwise the lack of a Cys-containing
`repeat might cause misfolding of the protein resulting
`in accumulation of TG in the ER and proteolysis.
`
`iodinated proteins and breakdown
`‘Abnormal’
`products It is known that besides TG other proteins
`such as albumin can be iodinated.
`Initial reports
`suggested that albumin was produced by the thyroid
`(49). In contrast with this, RT-PCR and labelling experi-
`ments showed that albumin in the thyroid gland origi-
`nates from blood (50) and is iodinated. Some of the
`iodinated albumin rapidly leaks out, giving a high
`protein bound iodine value in the blood circulation
`(51). Some is broken down by lysosomal enzymes in
`the thyroid, giving rise to the formation of iodinated
`peptides and iodinated histidine which are excreted in
`urine, providing a new criterion in the diagnosis of
`TG synthesis defects, even prenatally (52).
`Serum albumin may be taken up by the thyroid
`gland either by diffusion via the tight junctions or via
`basolateral endocytosis (53), and is iodinated in the
`follicular lumen. For the release of iodo-albumin into
`the circulation intra- or intercellular routes are postu-
`lated. The intracellular route follows the internalisation
`of iodinated albumin by pinocytosis and exocytosis at
`the basolateral membrane. The intercellular pathway
`is by leakage via the tight junctions.
`the
`The immuno-histological pictures show that
`inflow and outflow of protein under these pathological
`conditions occur mainly intercellularly (Fig. 3) and is
`strongly TSH dependent. In accordance with these find-
`ings we have shown that the width and number of
`chains of the tight junctions are inversely related to
`the TSH level, most likely resulting in increased protein
`leakage at higher TSH levels (54) (Fig. 4A, B).
`
`Iodide organification
`
`The oxidation and binding of iodine to tyrosine residues
`in TG are commonly referred to as iodide organifica-
`tion. Under normal conditions, the iodide uptake is
`the rate-limiting step in the iodination process. Iodide
`in the thyroid is derived from two distinct sources:
`iodide transported into the cell via a transporter
`system (NIS) (15) and iodide obtained via deiodination
`of organic iodine compounds within the gland. How
`far both iodide pools are exchangeable is a subject of
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`Figure 2 TG mutations in patients described in the literature, causing congenital hypothyroidism. WT, wild type; sp, signal peptide; "O
`arrow with solid tail, human pathology; "O
`- arrow with broken tail, animal pathology.
`
`controversy. However, in view of the rapid iodination
`reaction, catalysed by TPO,
`the free intracellular
`iodide concentration is low especially when compared
`with the total amount of glandular
`iodine. An
`impression of the thyroid iodide concentration in the
`thyroid cells can be obtained by the inhibition of
`iodide uptake after administration of sodium perchlor-
`ate or sodium thiocyanate, visualized by radioiodine
`(123I2). Normally no or , 10% of
`the radioiodine
`taken up will be released (Fig. 5).
`Thyroid peroxidase is 110 kDa in size, is encoded by
`the TPO gene (150 kb) containing 17 exons, and is
`located on chromosome 2p25. The gene is exclusively
`expressed in the thyroid gland under the influence
`of the thyroid transcription factors NKX2.1 (TTF1),
`FKL15 (TTF2) and PAX8. The protein shows one trans-
`membrane helix, with a large extracellular N-terminal
`part containing a haem group, essential for enzyme
`activity.
`The generation of hydrogen peroxide is a critical
`step in thyroid hormonogenesis. The thyroid oxidase
`1 (ThOX1) and 2 (ThOX2) genes have recently been
`identified as coding for components of the hydrogen
`peroxide generation system of the thyroid (55). The
`mRNAs coding for ThOX1 and 2 are 5369 and 6126
`nucleotides, coding for proteins of 1551 and 1548
`amino acids respectively. The structure of these pro-
`teins,
`inserted into the apical membrane,
`includes
`seven putative transmembrane domains, an NADPH
`
`Figure 3 Immuno-histochemical picture of a follicle that contains
`albumin. Albumin antigens are present between the epithelial cells
`from the base to the top. The epithelium at the left of the septum
`is flat and does not show transepithelial passage of serum
`proteins. Small amounts of albumin antigens are present in the
`follicular lumen (magnification 400£).
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`Defects in iodine pathways 253
`
`Figure 4 Dependence of width of tight junction on plasma TSH levels. (A) Electron microscopic pictures from freeze fracture sections
`from normal thyroid tissue and from goitre tissue (high TSH). (From Van Uyen et al. (54)). (B) Width of tight junctions in relation to
`plasma TSH values, given in mU/l. Each value is the average of about 300 measurements, the mean standard error is about 12 nm.
`TSH concentrations were determined by a special goat-bovine TSH radioimmunoassay (54).
`
`and a FAD binding site, two Ca2þ
`binding sites (EF
`hands) and haem binding sites. From reconstruction
`experiments, it is clear that more components have to
`be included in the thyroid oxidase system (56).
`
`Iodide organification defects
`
`Iodination defects can be partial or total. This depends
`on the degree to which iodide can be organified. Total
`organification defects (TIODs) are characterised by dis-
`charge of more than 90% of the (radio) iodide taken up
`by the gland within 1 hr after i.v. administration
`of sodium perchlorate (Fig. 5B). Partial organification
`defects (PIODs) are characterised by a discharge of more
`than 10% of the accumulated radioiodine (Fig. 5A).
`
`Partial iodide organification defects As iodination
`needs hydrogen peroxide, iodide and thyroglobulin as
`substrates at the outside of the apical membrane of
`the thyrocyte, a shortage in one of these components
`at the right place will give diminished iodination. A dis-
`order in the function of pendrin may cause a dimin-
`ished iodide transport over
`the apical membrane
`which results in iodide remaining in the thyrocyte. By
`inhibition of the inflow by perchlorate ions, a partial
`outflow will occur, resulting in a partial organification
`defect. In the case of a TG deficiency, insufficient protein
`may be present for the amount of iodide, occasionally
`resulting in a partial organification defect.
`Furthermore, partial
`iodide organification defects
`may be caused by a diminished organification of
`
`Figure 5 Radio-iodide uptake curves as observed in (A) partial iodide organification defects and (B) total iodide organification defects.
`2 ions stops the uptake or causes
`The grey area represents the normal uptake curves. Inhibition of the normal iodide uptake by ClO4
`a small decrease of , 10% of the iodide taken up. Partial iodide organification defect is defined as a wash-out of 10– 90%. When
`wash-out is . 90%, the defect is defined as total iodide organification defect.
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`EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) 149
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`iodide caused by disorders in thyroid peroxidase or thy-
`roid oxidase. Recently, a partial organification defect
`(perchlorate discharge 76 – 84%), caused by biallelic
`mutations in the TPO gene, has been reported. The
`three siblings, who were mildly hypothyroid, were
`found to be compound heterozygous for a missense
`mutation (G1687T) and a deletion in exon 10
`(1808 –1813del) (57). However, monoallelic mutations
`in the ThOX2 gene are found to cause partial organifi-
`cation defects, causing transient congenital hypothyr-
`oidism after birth (58). Obviously, thyroid hormone
`production is inadequate to fulfil the amount of thyrox-
`ine needed after birth and is most likely caused by too
`low a production of hydrogen peroxide. The regulation
`mechanism is not yet known.
`
`Total iodide organification defects Patients with
`severe congenital hypothyroidism, showing a total
`discharge (. 90%) of radio-iodine taken up by the
`thyroid after administration of sodium perchlorate
`within 1 hr, are classified under
`the category of
`total organification defect. Missense, nonsense, splice
`defects and frame shift mutations have been described
`by several groups (59, 60). From a Dutch study in
`45 patients from 35 families the molecular diagnosis
`was made (Table 3). The mutations found are: frame-
`shift mutations (six different ones) which occur most
`frequently: 39 of the 69 studied TPO alleles i.e. 57%,
`missense mutations (six different ones) found in 15
`mutated alleles (22%),
`followed by mutations that
`putatively affect splicing (three different ones) respon-
`sible for five affected alleles (7%) and finally one non-
`sense mutation that affects four TPO alleles (6%).
`The most
`frequently occurring single mutation is
`the GGCC duplication in exon 8 (61), at nucleotide
`position 1227, detected in 36% of the investigated
`TPO alleles. This mutation has been found in 51%
`of the Dutch TIOD families, either in a homozygous
`or in a compound heterozygous fashion.
`In one
`patient with severe congenital hypothyroidism and a
`classical
`total organification defect, a homozygous
`deletion (DT2512; codon 808) in exon 14 was ident-
`ified. The homozygosity in the patient was due to
`partial isodisomy of the short arm of chromosome 2
`carrying a defective TPO gene (Table 3). The patient,
`who was born small for gestational age, was healthy
`
`Table 3 Hereditary character of molecular defects in total iodide
`organification defects. Total number of families studied was 35.
`
`Hereditary character
`
`No. of families
`
`Homozygous for a mutation in TPO
`Compound heterozygous for mutations in TPO
`Isodisomy (chromosome 2p)
`Mutation in 1 allele of the TPO gene
`No mutations found in the cDNA coding for TPO
`
`13
`16
`1
`4*
`1
`
`* Mutations in promoter regions or intron sequences remain possible.
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`with normal growth and development while being
`treated with thyroxine. He has a normal phenotype
`except for a unilateral preauricular skin tag. This
`shows that partial maternal isodisomy (2pter – 2p12)
`is compatible with no or a minimal
`influence on
`normal development (62).
`In one patient showing severe congenital hypothy-
`roidism characterised by a total organification defect,
`no mutations were found in the gene coding for thyroid
`peroxidase (Table 3). In this case we found a bi-allelic
`mutation in the ThOX2 gene. These findings prove
`that the ThOX2 enzymatic activity is essential for thy-
`roidal hydrogen peroxide generation and that transient
`congenital hypothyroidism can be genetically deter-
`mined (58). From the results obtained, it is clear that
`total organification defects
`show an autosomal
`recessive inheritance pattern with an incidence of
`about 1:60 000 births mainly caused by defects in the
`TPO gene.
`
`Defects in recycling of iodine TG, internalised by
`(micro) pinocytosis from the follicular lumen, has
`been found to be present in the early and late endo-
`somes.
`In these organelles, containing proteolytic
`enzymes, thyroid hormones are freed. The hydrolysate
`contains amino acids,
`including mono- and di-
`iodotyrosine (MIT and DIT respectively). Subsequently,
`MIT and DIT are deiodinated by a specific dehalogenase
`found not only in the thyroid but also in some periphe-
`ral organs (63).
`Recently, the identification of a gene called DEHAL1
`from a thyroid SAGE library based on expression in
`thyroid, liver, kidney and mammary gland has been
`reported by our group (64). The main open reading
`frame encodes a novel human enzyme with a conserved
`nitroreductase domain that is FMN dependent and
`catalyses the dehalogenation of iodotyrosines. It is the
`first cloned vertebrate dehalogenase, providing the mol-
`ecular basis for an evolutionary conserved mechanism
`for iodine recycling.
`
`Conclusion
`
`In recent years much progress has been made in the
`clarification of thyroid defects at the molecular level,
`especially in the field of thyroid dyshormonogenesis. It
`might give the impression that most of the inherited
`defects in thyroid dyshormonogenesis have been eluci-
`dated. However, in a large number of patients the thy-
`roid defect cannot be diagnosed at the molecular level.
`Moreover, results of thyroid SAGE analyses (65) show
`that about 70% of the expressed transcripts could not
`be linked to known genes. From this, it is clear that
`there is still much to be investigated in order to eluci-
`date the mechanism of thyroid hormonogenesis and
`its associated disorders.
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`Defects in iodine pathways 255
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`Acknowledgements
`
`I wish to thank all members of the department of Pae-
`diatric Endocrinology for their contribution to these
`investigations. I thank the groups of Vassart/Dumont
`(Universite´ Libre, Brussels), Gru¨ ters/Krude (Humbold
`University, Berlin), Van Dijk (Ve