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Genetics and phenomics of hypothyroidism and goiter due to TPO mutations
Molecular and Cellular Endocrinology 322 (2010) 38–43
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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Review
Genetics and phenomics of hypothyroidism and goiter due to TPO mutations Carrie Ris-Stalpers a,∗ , Hennie Bikker b a b
Laboratory for Reproductive Biology, Academic Medical Center G2-133, PO Box 22700, 1100 DE Amsterdam, The Netherlands Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history: Received 26 June 2009 Received in revised form 5 February 2010 Accepted 5 February 2010 Keywords: Thyroid Peroxidase Hypothyroidism Iodine Iodide
a b s t r a c t Thyroid peroxidase (TPO) is a heme binding protein localized on the apical membrane of the thyrocyte. TPO enzymatic activity is essential for thyroid hormonogenesis. Inactivating mutations form the molecular basis for a specific subtype of congenital hypothyroidism: thyroid dyshormonogenesis due to an iodide organification defect. The most common phenotype of this autosomal recessive disease is a total iodide organification defect, with severe and permanent hypothyroidism as a consequence. Currently 61 properly annotated mutations in the TPO gene have been reported, of which the majority are missense mutations. Functional data of most missense mutations is not available, making it necessary to revert to in silico methods for functional interpretation of mutations. We hypothesize that iodine status is the main phenomic modifier of TPO function. © 2010 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5.
The function of TPO in thyroid physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The TPO gene, mRNA, and protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of TPO in hypothyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The phenomics of TPO mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A.Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. The function of TPO in thyroid physiology Thyroid peroxidase or thyroperoxidase (TPO) (OMIM 606765) is a glycosylated membrane bound hemoprotein localized on the apical membrane of the thyrocyte where it plays an essential role in the process of thyroid hormone synthesis. The thyrocytes that surround the follicular lumen are the thyroid hormone forming units within the thyroid gland. Fig. 1 (top part) represents a schematically drawn thyrocyte, highlighting the main proteins essential to thyroid hormone synthesis, with special focus on the iodination and coupling process.
Abbreviations: CH, congenital hypothyroidism; DUOXA2, DUOX2 maturation factor; DUOX2, dual oxidase 2; IYD, iodotyrosines deiodinase; SLC5A5, sodium/iodide symporter; SLC26A4, pendrin; TG, thyroglobulin; PIOD, partial iodide organification defect; TIOD, total iodide organification defect; TPO, thyroperoxidase/thyroid peroxidase. ∗ Corresponding author. Tel.: +31 20 566 5625. E-mail address: [email protected] (C. Ris-Stalpers). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.02.008
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The rare element iodine is an indispensable component of thyroid hormone and needs to be ingested through the diet. Ingested iodine is reduced to iodide and adsorbed in the small intestine after which it is transported in the plasma. Thyroid hormone synthesis cannot take place unless adequate amounts of iodide are taken up from the plasma across the basal membrane of the thyrocyte and are subsequently deposited at the apical membrane. At least two proteins are involved in this process; the sodium-iodide symporter (NIS or SLC5A5) at the basal membrane (Dai et al., 1996) and at the apical membrane iodide is effluxed by pendrin (SLC26A4) (Yoshida et al., 2002). At the apical membrane in the follicular lumen, proteins, enzymes, and elements necessary for thyroid hormone synthesis are in close contact. Thyroglobulin (TG)(Cody, 1984; van de Graaf et al., 2001) is the scaffold protein for thyroid hormone synthesis. TG contains tyrosine residues that can be iodinated by the oxidized form of iodide thus forming mono- or diiodotyrosine. For this oxidization of iodide (apart from iodide itself), H2 O2 is required as the final electron acceptor. The H2 O2 generating system is formed
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Fig. 1. Schematic representation of a thyrocyte, highlighting the process of thyroid hormonogenesis (top part). The steps in hormonogenesis in which TPO plays a role are depicted in the bottom part (based on Physiology and Pathophysiology of the DUOXes, 2006. Antioxid. Redox. Signal. 8, 1563–1572, Fig. 2, with permission).
by DUOX2 (De Deken et al., 2000). For functional maturation of DUOX2, the ER-resident transmembrane DUOX2 maturation factor protein DUOXA2 is essential (Grasberger and Refetoff, 2006; Zamproni et al., 2008). The subsequent inter- or intrachain coupling between either two diiodotyrosine residues, or less efficiently between a diiodotyrosine and a monoiodotyrosine residue, results in the formation
of thyroxin (T4 ) or triiodothyronine (T3 ). Mono- and diiodotyrosine residues that are not coupled to form thyroid hormone, can be deiodinated by iodotyrosines deiodinase (IYD). The liberated iodine forms an intrathyroidal supply of iodide that can be reused for thyroid hormone synthesis (Afink et al., 2008; Gnidehou et al., 2006; Moreno et al., 2008). In the coupling process, the tyrosyl residue that contributes the iodophenolic inner ring is termed the acceptor
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site, while the residue that contributes the iodinated outer ring is termed the donor site. Both the iodination and coupling function (Fig. 1, bottom part) are classically attributed to TPO (Taurog, 1970) and the enzymatic activity of TPO is dependent on the association with a heme group. Intriguingly, the 500 most N-terminal amino acids of DUOX2 show 43% homology with TPO. The role of this peroxidase domain is currently not clear but it has been demonstrated that a DUOX fragment containing the TPO-like domain expressed in bacteria displays peroxidase activity (Edens et al., 2001). This work implies that the peroxidase domain of DUOX might also be able to cross-link iodinated tyrosine residues in thyroglobulin, putatively reducing the contribution of TPO in the process of thyroid hormonogenesis to the organification of iodide. These findings are in line with earlier work that, because T4 can be formed nonenzymatically, proposed that TPO is not necessarily required for the coupling of iodotyrosines (DeCrombrugghe et al., 1967). The essential role of TPO in the iodination of tyrosine residues (the organification process), however, is not disputed. Additionally, in studies with purified TPO H2 O2 is usually generated by the system glucose–glucoseoxidase, a flavoprotein enzyme that in the reduced form directly reacts with O2 to form H2 O2 . Within this experimental system (Bikker et al., 1997), only the iodination potential of TPO is measured.
2. The TPO gene, mRNA, and protein The TPO enzymatic activity has been described decades ago, and already at that time TPO was linked to the most frequent type of hereditary thyroid hormone synthesis defects (DeGroot and Niepomniszcze, 1977). Several groups reported the complete sequence of the human TPO coding region; a major full-length transcript consisting of 3048 nucleotides encoding 933 amino acids (GenBank accession number NM 000547.4) and a minor transcript lacking 171 nucleotides (Kimura et al., 1987; Libert et al., 1987; Magnusson et al., 1987). Shortly after, the complete structure of the TPO gene was elucidated demonstrating the presence of 16 coding exons (Kimura et al., 1989). The gene was mapped to chromosome 2p25 and covers about 150 kb of DNA (Abramowicz et al., 1990; de Vijlder et al., 1988; Kimura et al., 1989). The definition of exon/intron boundaries established that the alternative transcript lacks the 9th coding exon of the gene encoding amino acids 533–589. There is ample evidence that this second TPO transcript is not enzymatically active. Firstly, it encodes a protein that is not able to bind heme, an intrinsic feature of the enzymatic activity (Niccoli et al., 1997). Secondly, subjects homozygous for the inactivating TPO mutation Arg540X have reduced TPO mRNA levels that mainly consist of the exon 9 lacking transcript. In thyroid tissue of these patients, no enzymatic TPO activity was detected, although the presence of the exon 9 lacking transcript was demonstrated (Bikker et al., 1996). Additional minor splice variants have been described but their biological natures have not been determined. The overall process of thyroid hormone synthesis is stimulated by the pituitary hormone TSH that acts through the G-protein coupled TSH receptor (TSH-R) located on the basal membrane of the thyrocytes (Fig. 1, top part). Chronic TSH stimulation increases the amount of TPO and its targeting to the apical membrane (Penel et al., 1998). It has also been reported that the human TPO promoter is cAMP and thyrotropin responsive (Abramowicz et al., 1990) but this is in contrast to studies showing that transcription of the TPO gene is not regulated by TSH and cAMP (Foti et al., 1990). TSH-R signalling through other members of the G-protein family and the inositol phosphatise/Ca2+ cascade may also contribute to the regulation of TPO expression (Buch et al., 2008; Grasberger et al., 2007; Laugwitz et al., 1996).
It is clearly established that transcription of the TPO gene is under control of the transcription factors NKX2-1 (TTF1), FOXE1 (TTF2) and PAX8 and that there are binding sites for these transcription factors present in the TPO promoter. Especially PAX 8 seems to be important for tissue-specific TPO expression (Esposito et al., 1998).
3. The role of TPO in hypothyroidism Congenital hypothyroidism is the most common endocrine disease, affecting 1 in every 1200 newborns in the Netherlands (Vulsma and de Vijlder, 2002). Defects in the process of thyroid hormone synthesis account for about 15% of all cases of permanent congenital hypothyroidism in the Netherlands (de Vijlder et al., 1997). If untreated, hypothyroidism due to dyshormonogenesis leads to increased TSH secretion, thyroid stimulation, and goiter in an attempt to compensate for the diminished capacity of the thyroid gland to produce thyroid hormone. Because thyroid hormone is essential for brain development during fetal and early postnatal life, untreated hypothyroidism will cause both cognitive and motor deficits. The severity of these neurodevelopmental problems will depend on the severity and duration of the hypothyroid state (Hulse, 1984). In patients suspect of a complete or partial TPO deficiency, TPO enzymatic activity is evaluated in vivo by a radioiodine uptake and perchlorate (ClO4 − ) discharge test. Because of elevated TSH levels, patients show a very rapid initial uptake of radioiodine that reaches a maximum within 30 min. Subsequent administration of ClO4 − competitively inhibits thyroidal iodide uptake by the sodium/iodide symporter SLC5A5. In case of unaffected iodination and coupling, the radioiodine taken up by the thyroid is protein bound (organified) and less than 10% is washed out. In case of a total iodide organification defect (TIOD), over 90% is discharged from the intrathyroidal iodide pool, indicating that the iodide that is taken up was not incorporated into protein. Discharge levels between 10 and 90% are termed partial iodide organification defects (PIOD) (de Vijlder and Vulsma, 1996). In a recent study of 183 infants with congenital hypothyroidism, 13.7% of patients was diagnosed with an iodine organification defect (Cavarzere et al., 2008). Patients displaying iodide organification defects were already described in the second half of the 20th century when radioisotopes became available (Djemli et al., 2006). Currently there are two genotypes explaining TIOD, while the molecular basis of PIOD is more diverse. The established molecular basis of TIOD is either due to bi-allelic inactivation mutations in the TPO gene, or bi-allelic inactivation mutations in the DUOX2 gene. Bi-allelic inactivation of DUOX2 results in a complete block of H2 O2 generation. This is a relatively rare genotype of which currently only one case has been reported (Moreno et al., 2002). Inactivating mutations in the TPO gene resulting in a TIOD are much more frequent (see supplementary Table 1, Fig. 2). The genetic basis for a partial iodide organification defect is the other way round: several DUOX2 (Grasberger, this issue) mutations and only rarely TPO mutations have been reported. Inactivating mono-allelic DUOX2 mutations have also been reported in relation to transient forms of relatively mild hypothyroidism (Moreno et al., 2002). Additionally, mutations in DUOXA2 (Zamproni et al., 2008) and SLC26A4 have been reported as the molecular basis for PIOD. SCL26A4 mutations are causative for Pendred syndrome, a combination of sensorineural hearing loss and a PIOD, with varying degrees of severity in both clinical features (Kopp, this issue). PIOD is also sometimes seen in patients with a putative thyroglobulin synthesis defect (Hishinuma et al., 1999; Rivolta and
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Fig. 2. Schematic drawing of the TPO gene with all the annotated mutations reported to date and present in Supplementary Table 1. Exons are drawn to scale, intron sequences are not. Mutations are depicted on top of exons, total number of mutations per exon are indicated below. Exon 7, 8 and 9 encode the heme binding catalytic site of the TPO protein.
Targovnik, 2006), probably caused by inadequate iodide binding capacity of TG molecules with an aberrant structure or impaired intracellular transport, posttranslational processing and reduced secretion rate of mutated and misfolded TG molecules. The marked phenotypic variability observed in PIOD patients probably reflects the diversity in underlying genetic abnormalities (Cavarzere et al., 2008). The accepted term for thyroid peroxidase deficiency in Online Mendelian Inheritance in Man database is genetic defects in thyroid hormonogenesis 2A (OMIM # 274500). TPO defects are commonly inherited in an autosomal recessive fashion (Park and Chatterjee, 2005). Since the first report in 1992 (Abramowicz et al., 1992) currently 67 different mutations are reported in the Human Gene Mutation Database (status June 18th, 2009) (http://portal.biobaseinternational.com/hgmd/pro/). Re-evaluating the corresponding literature identified 61 properly annotated mutations in the TPO gene. The majority of mutations are localized in exons 7, 8 and 9 of the gene, encoding the catalytic heme binding domain of the protein (Fig. 2). There are 13 frameshift mutations reported, 12 of which will probably lead to nonsense mediated mRNA decay (NMD) (Shyu et al., 2008). Three mutations affect splicing; two because they involve the donor or acceptor splice consensus site, and one based on in silico analysis. Six nonsense mutations have been reported and are also expected to result in NMD and there are two small in frame deletions. The majority (37 out of 61) are missense mutations, of which five nucleotide substitutions putatively affect splicing (Supplementary Table 1). Although functional analysis of missense mutations is important, it is usually not feasible. There are several in silico possibilities to evaluate putative functional effects of missense mutations included and some data are reported in Supplementary Table 1. SIFT (Ng and Henikoff, 2003) and PolyPhen (Ramensky et al., 2002) were used to predict whether an amino acid substitution affects protein function
based on sequence homology and the physical properties of the amino acids involved. Additionally the level of cross species conservation of the mutated amino acid was determined (Siepel et al., 2005). Based on these analyses, there are several missense mutations in exon 7 of which the evidence for the inactivating nature of the mutation is unconvincing. A legitimate hypothesis would be that obligatory heterozygous carriers (for instance parents of children with bi-allelic inactivating TPO mutations and a TIOD phenotype) might suffer from PIOD. This has however never been reported. There have been descriptions of single TPO-mutated alleles in TIOD patients, but more detailed genetic analysis could explain this by mono-allelic expression of the mutant TPO allele in thyroid tissue not caused by major deletions of the wild type allele (Fugazzola et al., 2003) or co-segregation of a mutated allele with a null allele (Kotani et al., 2001). It is usually assumed that inactivating TPO mutations always lead to TIOD, but recent reports indicate that some patients with less severe inactivating mutations display PIOD (Kotani et al., 2003; Nascimento et al., 2003). Fig. 3 provides a short algorithm describing the best rational to identify a genotype causing a specific phenotype, based on currently available literature. 4. The phenomics of TPO mutations Iodine is the main environmental factor interacting with TPO. Excess iodide is known to acutely inhibit thyroid hormone synthesis in case of normally functioning thyroid gland (The Wolff–Chaikoff effect) (Wolff and Chaikoff, 1948). The proposed mechanism is that excess iodide leads to the formation of 2iodohexadecanal that inhibits H2 O2 generation thereby inhibiting the TPO catalyzed iodination (Panneels et al., 1994).
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Fig. 3. Algorithm. Algorithm in aid of the elucidation of the molecular basis of iodide organification defects. In black decisions to be made based on literature evidence. In grey, at the moment hypothetical options for the molecular basis of iodide organification defects. In case of iodide excess, diagnostics should be repeated at a later date to evaluate thyroid function under adequate iodine status.
One patient who presented with congenital hypothyroidism was diagnosed as having a TIOD based on over 90% discharge in the perchlorate-discharge test. Hypothyroidism turned out to be of a transient nature which is highly unusual in case of TIOD. This patient probably suffered from a fetal iodine overload caused by maternal use of iodinated disinfectant (Cavarzere et al., 2008). It is tempting to speculate that iodine status could modify the phenotype of TPO mutations. There are however no data to substantiate this. The routine measurement of urinary iodine excretion in patients with CH due to an iodide organification defect might confirm this hypothesis (Fig. 3).
of tyrosine residues or also in the coupling of iodinated tyrosine residues to thyroid hormone, is a matter of dispute. Inactivating mutations in TPO are the main cause for autosomal recessive iodide organification defects, with currently 61 properly annotated mutations reported. It is temping to speculate that combined variations in different proteins involved in iodination and/or coupling might also provide a molecular basis for congenital hypothyroidism and iodide organification defects. TPO enzymatic activity is highly dependent on adequate levels of the rare element iodine. Therefore it is likely that the iodine status is a major determinant in the phenomics of iodide organification defects.
5. Conclusion Appendix A. Supplementary data Activity of the glycosylated membrane bound hemoprotein TPO is essential for thyroid hormone synthesis at the apical membrane of the thyrocyte. Whether TPO is involved only in the iodination
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2010.02.008.
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Human thyroperoxidase in its alternatively spliced form (TPO2) is enzymatically inactive and exhibits changes in intracellular processing and trafficking. J. Biol. Chem. 272 (47), 29487–29492. Panneels, V., Van den Bergen, H., Jacoby, C., Braekman, J.C., Van Sande, J., Dumont, J.E., Boeynaems, J.M., 1994. Inhibition of H2 O2 production by iodoaldehydes in cultured dog thyroid cells. Mol. Cell. Endocrinol. 102 (1–2), 167–176. Park, S.M., Chatterjee, V.K., 2005. Genetics of congenital hypothyroidism. J. Med. Genet. 42 (5), 379–389. Penel, C., Gruffat, D., Alquier, C., Benoliel, A.M., Chabaud, O., 1998. Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: a quantitative confocal microscopic study. J. Cell. Physiol. 174 (2), 160–169. Ramensky, V., Bork, P., Sunyaev, S., 2002. Human non-synonymous SNPs: server and survey. Nucleic Acids Res. 30 (17), 3894–3900. Rivolta, C.M., Targovnik, H.M., 2006. Molecular advances in thyroglobulin disorders. Clin. Chim. Acta 374 (1–2), 8–24. Shyu, A.B., Wilkinson, M.F., van, H.A., 2008. Messenger RNA regulation: to translate or to degrade. EMBO J. 27 (3), 471–481. Siepel, A., Bejerano, G., Pedersen, J.S., Hinrichs, A.S., Hou, M., Rosenbloom, K., Clawson, H., Spieth, J., Hillier, L.W., Richards, S., Weinstock, G.M., Wilson, R.K., Gibbs, R.A., Kent, W.J., Miller, W., Haussler, D., 2005. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15 (8), 1034–1050. Taurog, A., 1970. Thyroid peroxidase and thyroxine biosynthesis. Recent Prog. Horm. Res. 26, 189–247. van de Graaf, S.A., Ris-Stalpers, C., Pauws, E., Mendive, F.M., Targovnik, H.M., de Vijlder, J.J., 2001. Up to date with human thyroglobulin. J. Endocrinol. 170 (2), 307–321. Vulsma, T., de Vijlder, J.J.M., 2002. Thyroid disease in newborns, infants and children. In: Wass, J.A., Shalet, S.M. (Eds.), Oxford Textbook of Endocrinology and Diabetes. Oxford University Press, pp. 532–544. 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Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Review
Genetics and phenomics of hypothyroidism and goiter due to TPO mutations Carrie Ris-Stalpers a,∗ , Hennie Bikker b a b
Laboratory for Reproductive Biology, Academic Medical Center G2-133, PO Box 22700, 1100 DE Amsterdam, The Netherlands Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history: Received 26 June 2009 Received in revised form 5 February 2010 Accepted 5 February 2010 Keywords: Thyroid Peroxidase Hypothyroidism Iodine Iodide
a b s t r a c t Thyroid peroxidase (TPO) is a heme binding protein localized on the apical membrane of the thyrocyte. TPO enzymatic activity is essential for thyroid hormonogenesis. Inactivating mutations form the molecular basis for a specific subtype of congenital hypothyroidism: thyroid dyshormonogenesis due to an iodide organification defect. The most common phenotype of this autosomal recessive disease is a total iodide organification defect, with severe and permanent hypothyroidism as a consequence. Currently 61 properly annotated mutations in the TPO gene have been reported, of which the majority are missense mutations. Functional data of most missense mutations is not available, making it necessary to revert to in silico methods for functional interpretation of mutations. We hypothesize that iodine status is the main phenomic modifier of TPO function. © 2010 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5.
The function of TPO in thyroid physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The TPO gene, mRNA, and protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of TPO in hypothyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The phenomics of TPO mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A.Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. The function of TPO in thyroid physiology Thyroid peroxidase or thyroperoxidase (TPO) (OMIM 606765) is a glycosylated membrane bound hemoprotein localized on the apical membrane of the thyrocyte where it plays an essential role in the process of thyroid hormone synthesis. The thyrocytes that surround the follicular lumen are the thyroid hormone forming units within the thyroid gland. Fig. 1 (top part) represents a schematically drawn thyrocyte, highlighting the main proteins essential to thyroid hormone synthesis, with special focus on the iodination and coupling process.
Abbreviations: CH, congenital hypothyroidism; DUOXA2, DUOX2 maturation factor; DUOX2, dual oxidase 2; IYD, iodotyrosines deiodinase; SLC5A5, sodium/iodide symporter; SLC26A4, pendrin; TG, thyroglobulin; PIOD, partial iodide organification defect; TIOD, total iodide organification defect; TPO, thyroperoxidase/thyroid peroxidase. ∗ Corresponding author. Tel.: +31 20 566 5625. E-mail address: [email protected] (C. Ris-Stalpers). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.02.008
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The rare element iodine is an indispensable component of thyroid hormone and needs to be ingested through the diet. Ingested iodine is reduced to iodide and adsorbed in the small intestine after which it is transported in the plasma. Thyroid hormone synthesis cannot take place unless adequate amounts of iodide are taken up from the plasma across the basal membrane of the thyrocyte and are subsequently deposited at the apical membrane. At least two proteins are involved in this process; the sodium-iodide symporter (NIS or SLC5A5) at the basal membrane (Dai et al., 1996) and at the apical membrane iodide is effluxed by pendrin (SLC26A4) (Yoshida et al., 2002). At the apical membrane in the follicular lumen, proteins, enzymes, and elements necessary for thyroid hormone synthesis are in close contact. Thyroglobulin (TG)(Cody, 1984; van de Graaf et al., 2001) is the scaffold protein for thyroid hormone synthesis. TG contains tyrosine residues that can be iodinated by the oxidized form of iodide thus forming mono- or diiodotyrosine. For this oxidization of iodide (apart from iodide itself), H2 O2 is required as the final electron acceptor. The H2 O2 generating system is formed
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Fig. 1. Schematic representation of a thyrocyte, highlighting the process of thyroid hormonogenesis (top part). The steps in hormonogenesis in which TPO plays a role are depicted in the bottom part (based on Physiology and Pathophysiology of the DUOXes, 2006. Antioxid. Redox. Signal. 8, 1563–1572, Fig. 2, with permission).
by DUOX2 (De Deken et al., 2000). For functional maturation of DUOX2, the ER-resident transmembrane DUOX2 maturation factor protein DUOXA2 is essential (Grasberger and Refetoff, 2006; Zamproni et al., 2008). The subsequent inter- or intrachain coupling between either two diiodotyrosine residues, or less efficiently between a diiodotyrosine and a monoiodotyrosine residue, results in the formation
of thyroxin (T4 ) or triiodothyronine (T3 ). Mono- and diiodotyrosine residues that are not coupled to form thyroid hormone, can be deiodinated by iodotyrosines deiodinase (IYD). The liberated iodine forms an intrathyroidal supply of iodide that can be reused for thyroid hormone synthesis (Afink et al., 2008; Gnidehou et al., 2006; Moreno et al., 2008). In the coupling process, the tyrosyl residue that contributes the iodophenolic inner ring is termed the acceptor
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site, while the residue that contributes the iodinated outer ring is termed the donor site. Both the iodination and coupling function (Fig. 1, bottom part) are classically attributed to TPO (Taurog, 1970) and the enzymatic activity of TPO is dependent on the association with a heme group. Intriguingly, the 500 most N-terminal amino acids of DUOX2 show 43% homology with TPO. The role of this peroxidase domain is currently not clear but it has been demonstrated that a DUOX fragment containing the TPO-like domain expressed in bacteria displays peroxidase activity (Edens et al., 2001). This work implies that the peroxidase domain of DUOX might also be able to cross-link iodinated tyrosine residues in thyroglobulin, putatively reducing the contribution of TPO in the process of thyroid hormonogenesis to the organification of iodide. These findings are in line with earlier work that, because T4 can be formed nonenzymatically, proposed that TPO is not necessarily required for the coupling of iodotyrosines (DeCrombrugghe et al., 1967). The essential role of TPO in the iodination of tyrosine residues (the organification process), however, is not disputed. Additionally, in studies with purified TPO H2 O2 is usually generated by the system glucose–glucoseoxidase, a flavoprotein enzyme that in the reduced form directly reacts with O2 to form H2 O2 . Within this experimental system (Bikker et al., 1997), only the iodination potential of TPO is measured.
2. The TPO gene, mRNA, and protein The TPO enzymatic activity has been described decades ago, and already at that time TPO was linked to the most frequent type of hereditary thyroid hormone synthesis defects (DeGroot and Niepomniszcze, 1977). Several groups reported the complete sequence of the human TPO coding region; a major full-length transcript consisting of 3048 nucleotides encoding 933 amino acids (GenBank accession number NM 000547.4) and a minor transcript lacking 171 nucleotides (Kimura et al., 1987; Libert et al., 1987; Magnusson et al., 1987). Shortly after, the complete structure of the TPO gene was elucidated demonstrating the presence of 16 coding exons (Kimura et al., 1989). The gene was mapped to chromosome 2p25 and covers about 150 kb of DNA (Abramowicz et al., 1990; de Vijlder et al., 1988; Kimura et al., 1989). The definition of exon/intron boundaries established that the alternative transcript lacks the 9th coding exon of the gene encoding amino acids 533–589. There is ample evidence that this second TPO transcript is not enzymatically active. Firstly, it encodes a protein that is not able to bind heme, an intrinsic feature of the enzymatic activity (Niccoli et al., 1997). Secondly, subjects homozygous for the inactivating TPO mutation Arg540X have reduced TPO mRNA levels that mainly consist of the exon 9 lacking transcript. In thyroid tissue of these patients, no enzymatic TPO activity was detected, although the presence of the exon 9 lacking transcript was demonstrated (Bikker et al., 1996). Additional minor splice variants have been described but their biological natures have not been determined. The overall process of thyroid hormone synthesis is stimulated by the pituitary hormone TSH that acts through the G-protein coupled TSH receptor (TSH-R) located on the basal membrane of the thyrocytes (Fig. 1, top part). Chronic TSH stimulation increases the amount of TPO and its targeting to the apical membrane (Penel et al., 1998). It has also been reported that the human TPO promoter is cAMP and thyrotropin responsive (Abramowicz et al., 1990) but this is in contrast to studies showing that transcription of the TPO gene is not regulated by TSH and cAMP (Foti et al., 1990). TSH-R signalling through other members of the G-protein family and the inositol phosphatise/Ca2+ cascade may also contribute to the regulation of TPO expression (Buch et al., 2008; Grasberger et al., 2007; Laugwitz et al., 1996).
It is clearly established that transcription of the TPO gene is under control of the transcription factors NKX2-1 (TTF1), FOXE1 (TTF2) and PAX8 and that there are binding sites for these transcription factors present in the TPO promoter. Especially PAX 8 seems to be important for tissue-specific TPO expression (Esposito et al., 1998).
3. The role of TPO in hypothyroidism Congenital hypothyroidism is the most common endocrine disease, affecting 1 in every 1200 newborns in the Netherlands (Vulsma and de Vijlder, 2002). Defects in the process of thyroid hormone synthesis account for about 15% of all cases of permanent congenital hypothyroidism in the Netherlands (de Vijlder et al., 1997). If untreated, hypothyroidism due to dyshormonogenesis leads to increased TSH secretion, thyroid stimulation, and goiter in an attempt to compensate for the diminished capacity of the thyroid gland to produce thyroid hormone. Because thyroid hormone is essential for brain development during fetal and early postnatal life, untreated hypothyroidism will cause both cognitive and motor deficits. The severity of these neurodevelopmental problems will depend on the severity and duration of the hypothyroid state (Hulse, 1984). In patients suspect of a complete or partial TPO deficiency, TPO enzymatic activity is evaluated in vivo by a radioiodine uptake and perchlorate (ClO4 − ) discharge test. Because of elevated TSH levels, patients show a very rapid initial uptake of radioiodine that reaches a maximum within 30 min. Subsequent administration of ClO4 − competitively inhibits thyroidal iodide uptake by the sodium/iodide symporter SLC5A5. In case of unaffected iodination and coupling, the radioiodine taken up by the thyroid is protein bound (organified) and less than 10% is washed out. In case of a total iodide organification defect (TIOD), over 90% is discharged from the intrathyroidal iodide pool, indicating that the iodide that is taken up was not incorporated into protein. Discharge levels between 10 and 90% are termed partial iodide organification defects (PIOD) (de Vijlder and Vulsma, 1996). In a recent study of 183 infants with congenital hypothyroidism, 13.7% of patients was diagnosed with an iodine organification defect (Cavarzere et al., 2008). Patients displaying iodide organification defects were already described in the second half of the 20th century when radioisotopes became available (Djemli et al., 2006). Currently there are two genotypes explaining TIOD, while the molecular basis of PIOD is more diverse. The established molecular basis of TIOD is either due to bi-allelic inactivation mutations in the TPO gene, or bi-allelic inactivation mutations in the DUOX2 gene. Bi-allelic inactivation of DUOX2 results in a complete block of H2 O2 generation. This is a relatively rare genotype of which currently only one case has been reported (Moreno et al., 2002). Inactivating mutations in the TPO gene resulting in a TIOD are much more frequent (see supplementary Table 1, Fig. 2). The genetic basis for a partial iodide organification defect is the other way round: several DUOX2 (Grasberger, this issue) mutations and only rarely TPO mutations have been reported. Inactivating mono-allelic DUOX2 mutations have also been reported in relation to transient forms of relatively mild hypothyroidism (Moreno et al., 2002). Additionally, mutations in DUOXA2 (Zamproni et al., 2008) and SLC26A4 have been reported as the molecular basis for PIOD. SCL26A4 mutations are causative for Pendred syndrome, a combination of sensorineural hearing loss and a PIOD, with varying degrees of severity in both clinical features (Kopp, this issue). PIOD is also sometimes seen in patients with a putative thyroglobulin synthesis defect (Hishinuma et al., 1999; Rivolta and
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Fig. 2. Schematic drawing of the TPO gene with all the annotated mutations reported to date and present in Supplementary Table 1. Exons are drawn to scale, intron sequences are not. Mutations are depicted on top of exons, total number of mutations per exon are indicated below. Exon 7, 8 and 9 encode the heme binding catalytic site of the TPO protein.
Targovnik, 2006), probably caused by inadequate iodide binding capacity of TG molecules with an aberrant structure or impaired intracellular transport, posttranslational processing and reduced secretion rate of mutated and misfolded TG molecules. The marked phenotypic variability observed in PIOD patients probably reflects the diversity in underlying genetic abnormalities (Cavarzere et al., 2008). The accepted term for thyroid peroxidase deficiency in Online Mendelian Inheritance in Man database is genetic defects in thyroid hormonogenesis 2A (OMIM # 274500). TPO defects are commonly inherited in an autosomal recessive fashion (Park and Chatterjee, 2005). Since the first report in 1992 (Abramowicz et al., 1992) currently 67 different mutations are reported in the Human Gene Mutation Database (status June 18th, 2009) (http://portal.biobaseinternational.com/hgmd/pro/). Re-evaluating the corresponding literature identified 61 properly annotated mutations in the TPO gene. The majority of mutations are localized in exons 7, 8 and 9 of the gene, encoding the catalytic heme binding domain of the protein (Fig. 2). There are 13 frameshift mutations reported, 12 of which will probably lead to nonsense mediated mRNA decay (NMD) (Shyu et al., 2008). Three mutations affect splicing; two because they involve the donor or acceptor splice consensus site, and one based on in silico analysis. Six nonsense mutations have been reported and are also expected to result in NMD and there are two small in frame deletions. The majority (37 out of 61) are missense mutations, of which five nucleotide substitutions putatively affect splicing (Supplementary Table 1). Although functional analysis of missense mutations is important, it is usually not feasible. There are several in silico possibilities to evaluate putative functional effects of missense mutations included and some data are reported in Supplementary Table 1. SIFT (Ng and Henikoff, 2003) and PolyPhen (Ramensky et al., 2002) were used to predict whether an amino acid substitution affects protein function
based on sequence homology and the physical properties of the amino acids involved. Additionally the level of cross species conservation of the mutated amino acid was determined (Siepel et al., 2005). Based on these analyses, there are several missense mutations in exon 7 of which the evidence for the inactivating nature of the mutation is unconvincing. A legitimate hypothesis would be that obligatory heterozygous carriers (for instance parents of children with bi-allelic inactivating TPO mutations and a TIOD phenotype) might suffer from PIOD. This has however never been reported. There have been descriptions of single TPO-mutated alleles in TIOD patients, but more detailed genetic analysis could explain this by mono-allelic expression of the mutant TPO allele in thyroid tissue not caused by major deletions of the wild type allele (Fugazzola et al., 2003) or co-segregation of a mutated allele with a null allele (Kotani et al., 2001). It is usually assumed that inactivating TPO mutations always lead to TIOD, but recent reports indicate that some patients with less severe inactivating mutations display PIOD (Kotani et al., 2003; Nascimento et al., 2003). Fig. 3 provides a short algorithm describing the best rational to identify a genotype causing a specific phenotype, based on currently available literature. 4. The phenomics of TPO mutations Iodine is the main environmental factor interacting with TPO. Excess iodide is known to acutely inhibit thyroid hormone synthesis in case of normally functioning thyroid gland (The Wolff–Chaikoff effect) (Wolff and Chaikoff, 1948). The proposed mechanism is that excess iodide leads to the formation of 2iodohexadecanal that inhibits H2 O2 generation thereby inhibiting the TPO catalyzed iodination (Panneels et al., 1994).
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Fig. 3. Algorithm. Algorithm in aid of the elucidation of the molecular basis of iodide organification defects. In black decisions to be made based on literature evidence. In grey, at the moment hypothetical options for the molecular basis of iodide organification defects. In case of iodide excess, diagnostics should be repeated at a later date to evaluate thyroid function under adequate iodine status.
One patient who presented with congenital hypothyroidism was diagnosed as having a TIOD based on over 90% discharge in the perchlorate-discharge test. Hypothyroidism turned out to be of a transient nature which is highly unusual in case of TIOD. This patient probably suffered from a fetal iodine overload caused by maternal use of iodinated disinfectant (Cavarzere et al., 2008). It is tempting to speculate that iodine status could modify the phenotype of TPO mutations. There are however no data to substantiate this. The routine measurement of urinary iodine excretion in patients with CH due to an iodide organification defect might confirm this hypothesis (Fig. 3).
of tyrosine residues or also in the coupling of iodinated tyrosine residues to thyroid hormone, is a matter of dispute. Inactivating mutations in TPO are the main cause for autosomal recessive iodide organification defects, with currently 61 properly annotated mutations reported. It is temping to speculate that combined variations in different proteins involved in iodination and/or coupling might also provide a molecular basis for congenital hypothyroidism and iodide organification defects. TPO enzymatic activity is highly dependent on adequate levels of the rare element iodine. Therefore it is likely that the iodine status is a major determinant in the phenomics of iodide organification defects.
5. Conclusion Appendix A. Supplementary data Activity of the glycosylated membrane bound hemoprotein TPO is essential for thyroid hormone synthesis at the apical membrane of the thyrocyte. Whether TPO is involved only in the iodination
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2010.02.008.
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