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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-2276
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 2 627-633
Copyright © 2008 by The Endocrine Society

Pseudodominant Inheritance of Goitrous Congenital Hypothyroidism Caused by TPO Mutations: Molecular and in Silico Studies

Johnny Deladoëy, Nicole Pfarr, Jean-Marc Vuissoz, Jasmine Parma, Gilbert Vassart, Stefan Biesterfeld, Joachim Pohlenz and Guy Van Vliet

Endocrinology Service and Research Center (J.D., G.V.V.), Sainte-Justine Hospital and Department of Pediatrics, University of Montreal, Montreal, Canada H3T 1C5; Pediatric Endocrinology (N.P., J.P.), Department of Pediatrics, and Department of Pathology (S.B.), Children’s Hospital of Johannes Gutenberg University, D-55101 Mainz, Germany; Department of Pediatrics (J.-M.V.), University Children’s Hospital, University of Bern, CH-3010 Bern, Switzerland; and Interdisciplinary Research Institute for Human and Molecular Biology (J.P., G.V.), Faculty of Medicine, and Department of Genetics, Erasme Hospital, Free University of Brussels, B-1070 Brussels, Belgium

Address all correspondence and requests for reprints to: Guy Van Vliet, M.D., Hôpital Sainte-Justine, 3175 Côte Sainte-Catherine, Montréal, Québec, Canada H3T 1C5. E-mail: guy.van-vliet{at}recherche-ste-justine.qc.ca.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context and Objective: Most cases of goitrous congenital hypothyroidism (CH) from thyroid dyshormonogenesis 1) follow a recessive mode of inheritance and 2) are due to mutations in the thyroid peroxidase gene (TPO). We report the genetic mechanism underlying the apparently dominant inheritance of goitrous CH in a nonconsanguineous family of French Canadian origin.

Design, Setting, and Participants: Two brothers identified by newborn TSH screening had severe hypothyroidism and a goiter with increased 99mTc uptake. The mother was euthyroid, but the father and two paternal uncles had also been diagnosed with goitrous CH. After having excluded PAX8 gene mutations, we hypothesized that the underlying defect could be TPO mutations.

Results: Both compound heterozygous siblings had inherited a mutant TPO allele carried by their mother (c.1496delC; p.Pro499Argfs2X), and from their father, one brother had inherited a missense mutation (c.1978C->G; p.Gln660Glu) and the other an insertion (c.1955insT; p.Phe653Valfs15X). The thyroid gland of one uncle who is a compound heterozygote for TPO mutations (p.Phe653Valfs15X/p.Gln660Glu) was removed because of concurrent multiple endocrine neoplasia type 2A. Immunohistochemistry revealed normal TPO staining, implying that Gln660Glu TPO is expressed properly. Modeling of this mutant in silico suggests that its three-dimensional structure is conserved, whereas the electrostatic binding energy between the Gln660Glu TPO and its heme group becomes repulsive.

Conclusion: We report a pedigree presenting with pseudodominant goitrous CH due to segregation of three different TPO mutations. Although goitrous CH generally follows a recessive mode of inheritance, the high frequency of TPO mutations carriers may lead to pseudodominant inheritance.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Congenital primary hypothyroidism (CH) occurs in approximately 1 in 3000 live births (1). In 80% of cases, the disease is due to defects in early thyroid organogenesis resulting in either absent or ectopic thyroid glands (2). In this group, most cases appear to be nonfamilial, and the high discordance rate between monozygotic twins argues against simple Mendelian patterns of inheritance (3).

On the other hand, CH associated with an orthotopic goiter appears to follow a Mendelian pattern of inheritance. Most cases of goitrous CH with iodide organification defect are attributable to thyroid peroxidase (TPO) deficiency (4, 5, 6, 7). Other genes implicated in goitrous CH include those encoding thyroglobulin (Tg) (8), the sodium iodide symporter (NIS) (9), pendrin (PDS) (10), and thyroid oxidase 2 (THOX2) (11). The human TPO gene contains 17 exons and covers approximately 150 kb of chromosome 2p25. TPO is a heme-containing enzyme of 933 amino acids, which shares 42% sequence identity with human myeloperoxidase (MPO) (12, 13). The homology between TPO and MPO rises up to 74% in the vicinity of the heme group (14, 15). The mature enzyme is a membrane-linked homodimer (16). Absence of TPO activity implies the inability to iodinate tyrosine residues in thyroglobulin and to couple these residues to form thyroid hormones. To date, more than 50 mutations in the TPO gene have been described, resulting in a variable decrease in TPO bioactivity (OMIM access number *606755). Although most cases with CH caused by defective TPO follow a recessive mode of inheritance, some cases might be due either to monoallelic expression of the mutant allele (17) or to uniparental isodisomy for chromosome 2p (18). However, in these cases, the genetic defect is confined to one generation and is, therefore, not inherited.

We report a nonconsanguineous family of French Canadian origin presenting with an apparently dominant inheritance of thyroid dyshormonogenesis. Because of the dominant transmission of hypothyroidism with a gland in situ (19), we first sequenced the coding exons of the PAX8 gene directly from genomic DNA of the two affected boys and their affected father, but only polymorphisms were found. We next hypothesized that the euthyroid mother could be a carrier of a TPO mutation. This would explain the apparently dominant inheritance of the disease, a phenomenon defined as pseudodominance and well described in consanguineous families where the carrier rate is high (e.g. SHOX mutations in Langer mesomelic dysplasia) (20). Indeed, sequencing analysis of the TPO gene revealed that both probands had inherited a deletion from their mother (c.1496delC; p.Pro499Argfs2X: mutation in codon 499 and a frameshift ending with stop codon X at position 2 in shifted reading frame). From their father, who was compound heterozygous for TPO mutations, one brother had inherited a missense mutation (c.1978C->G; p.Gln660Glu) and the other an insertion (c.1955insT; p.Phe653Valfs15X). Two affected paternal uncles also had multiple endocrine neoplasia type 2A and had undergone thyroidectomy. The thyroid from one uncle was retrieved and studied by immunohistochemistry for TPO. Finally, to improve our understanding of structure-function relationships of the mutant protein, computational modeling was used to analyze the Gln660Glu TPO mutation.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study subjects

Written informed consent for genetic studies was given by adult individuals as well as by parents on behalf of their minor children. The two boys (III-1 and III-2) were diagnosed by the newborn screening program (III-1, TSH 282 mU/liter; III-2, TSH 453 mU/liter) on d 2 of life, and their thyroid scans showed enlarged thyroid glands in situ (see Fig. 1Go). Their father (II-3) had also been diagnosed by the newborn screening program on d 3 with a TSH level of 646 mU/liter. For the two boys and their father, L-T4 treatment was started immediately after diagnosis in the neonatal period, and they have had normal growth and development. The two affected uncles (subjects II-1 and II-2) were born before the implementation of the newborn screening program in Quebec and therefore were diagnosed later at ages 15 and 12 months, respectively, on the basis of growth retardation. Despite this late diagnosis, they appear to have normal intelligence.


Figure 1
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  		FIG. 1. Pseudodominant inheritance of congenital thyroid dyshormonogenesis. Results of thyroid function tests at screening and at diagnosis, clinical signs, thyroid position at scintigraphy, and TPO alleles are aligned with arrowed symbols in the pedigree. TPO alleles: A, c.1978C->G-p.Gln660Glu; B, c.1955insT-p.Phe653Valfs15X; C, c.1496delC-p.Pro499Argfs2X; WT, wild type.

 
There were also cases of multiple endocrine neoplasia type 2A in the paternal family. The two uncles (II-1 and II-2) carried the Cys634Phe mutation in RET, and they were thyroidectomized in their twenties. The paraffin-embedded thyroid gland of individual II-1 was retrieved and TPO expression assessed by immunohistochemistry. The father (II-3) of the index cases (III-1 and III-2) tested negative for RET mutations.

Molecular analysis

Genomic DNA was isolated from peripheral blood leukocytes by using the QIAamp Blood Kit (QIAGEN Inc., Mississauga, Ontario, Canada). The PAX8 and the TPO genes were amplified with primers flanking all coding exons as previously described (19, 21). The PCR products were then purified and sequenced directly using an automated sequencing system (3100Avant; Applied Biosystems, Darmstadt, Germany). Mutation nomenclature refers to the NCBI human TPO nucleotides sequence (NCBI access number NM_000547) and is expressed following the standard proposed by the Association for Molecular Pathology Training and Education Committee (22).

Immunohistochemistry

The thyroid gland from II-1 was obtained at surgery and paraffin embedded. TPO immunohistochemistry was performed using a mouse monoclonal antibody (MoAB47) according to the supplier’s protocol (Dako, Hamburg, Germany). After deparaffinization and rehydration, sections were subjected to a 15-min treatment in a steamer in Tris/EDTA buffer (pH 9.0) for antigen retrieval. The sections were incubated with primary antibody (1:100 dilution) in an autostainer (Dako Autostainer plus; Dako). As second antibody, an alkaline phosphatase-coupled antimouse antibody was used (Dako). The alkaline phosphatase complex was used with Red-K as chromogen (Dako), producing red staining. Finally, slides were counterstained with hematoxylin (Mayer’s hematoxylin; Dako) and mounted (see Fig. 3Go). Positive controls were made on sections of normal adult thyroid tissue (Fig. 3CGo). Negative controls were performed by omission of the primary antibody (Fig. 3DGo).


Figure 3
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  		FIG. 3. A and B, Immunohistochemistry of the thyroid from the affected uncle (II-1), stained with the anti-TPO antibody MoAB47 (x40) (A) and (x80) (B); C, thyroid from a healthy individual stained with MoAB47 (x80); D, negative control without MoAB47 (x60).

 
In silico study of the human TPO

The human TPO and MPO amino acid sequences were obtained from the Swiss-Prot database (http://expasy.org/sprot/). The sequence alignment showed an overall homology of 50% for amino acids 134–731 of TPO. This region, defined hereafter as MPO-like domain of TPO, encompassed the heme peroxidase domain. Next, the MODELLER 9v1 software package (23) was used 1) to generate a tertiary structure model of the MPO-like domain of the human TPO based upon the structure of human MPO (PDB accession no. 1d2v; 1.75-Å resolution), 2) to select the best model, and 3) to generate the mutant Gln660Glu TPO model for simulation. The quality of all selected models was assessed by the ProQ software, which is a neural-network-based method that extracts structural features, such as frequency of atoms-atoms contacts, to predict the quality of a model (http://www.sbc.su.se/~bjornw/ProQ/ProQ.cgi) (24). Then, we performed a molecular dynamics stimulation of 5 ns of the catalytic site of the TPO (a heme-protein complex) for the wild-type and the Gln660Glu TPO with the GROMACS 3.2.1 package (GROMOS force fields) (25). The first 4 nsec of the run was treated as a further equilibration simulation, and the remaining 1 nsec was saved and used for the analysis. After the stimulation, 1) mutant (and controls) were generated with MODELLER 9v1 from the wild-type to assess the impact of amino acid change on electrostatic forces, and 2) electrostatic binding energy of the heme-TPO (mutant and controls) interface was computed by solving the Poisson-Boltzmann equation with the continuum model APBS 0.5.0 (26) and compared with the concurrent value of the heme-wild-type TPO interface (i.e. relative electrostatic binding energy). This method has been shown to predict electrostatic binding energies reliably (26). All the pictures were produced with the The PyMOL Molecular Graphics System (2002) (http://www.pymol.org). Statistical analyses were performed as appropriate using the free statistical software R (27).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Molecular analysis of the PAX8 gene

PAX8 heterozygous mutations may have a wide phenotypic expression and, therefore, may be misdiagnosed initially as dyshormonogenesis (28). Thus, to exclude a rare form of dominant CH with orthotopic thyroid glands, we sequenced the PAX8 gene. Only two polymorphisms were found in the heterozygous state: rs 2084163 (a C/G transversion 104 nucleotides upstream of the initiator ATG) and c.*187 A->G (in the 3' untranslated segment, reference sequence NM_003466.3).

Molecular analysis of the TPO gene

To explain the pseudodominant inheritance in this pedigree, our hypothesis was that the mother was a carrier of a mutated TPO allele, which was transmitted to both siblings. To assess this, we sequenced the TPO gene in all family members. This revealed that 1) the mother was a carrier for a mutant TPO allele; 2) the two probands, the father and the uncle, were compound heterozygotes; and 3) all combinations of heterozygous mutations resulted in a similarly severe phenotype, implying that each mutation was responsible for a severe loss of function (see pedigree, Fig. 1Go). Indeed, both children (III-1 and III-2) had inherited a deletion from their mother (c.1496delC; p.Pro499Argfs2X). From their father, one brother (III-1) had inherited a transversion (c.1978C->G; p.Gln660Glu) and the other (III-2) an insertion (c.1955insT; p.Phe653Valfs15X). The 1978 G->C transversion in exon 11 results in the substitution of glutamic acid for glutamine at position 660 (Gln660Glu). This mutation has already been described in six Portuguese families as well as in two Brazilian families (7, 29). The two other mutations (c.1496delC and c.1955insT) are novel and lead to frameshifts with a premature termination signal at codon 501 for c.1496delC and at codon 668 for c.1995insT, both located in the heme peroxidase domain of the TPO (Fig. 2AGo). Finally, all the mutations found in this pedigree were absent in 50 normal individuals.


Figure 2
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  		FIG. 2. The TPO structure. A, Schematic view of the human TPO structure; plain arrows indicate the position of the identified mutations within the animal heme oxidase, and dashed arrow indicates the position of the MoAB47 epitope. B, Amino acids alignment across species in the vicinity of the Gln 660 performed with Clustal W 1.83 (http://www.ebi.ac.uk/clustalw/); Gln is abbreviated with the letter Q (arrow). C, Ribbon representation of the modeled structure of TPO; the Gln660 residue (arrow) and the heme are represented with sticks; the iron atom is represented as a blue sphere in the center of the heme.

 
Assessment of TPO expression by immunohistochemistry

To determine whether the mutant TPO is expressed properly, we performed immunohistochemistry on the thyroid gland of the affected uncle (II-1) who is compound heterozygous for Phe653Valfs15X/Gln660Glu. The frameshift causes a stop codon at position 668; thus, the mutant protein will not be recognized by MoAB47 because its epitope lies between residues 713–717 (30) (Fig. 2AGo). Therefore, the signal, especially at the apical membrane, can only be due to Gln660Glu TPO (Fig. 3Go, A and B). The cellular distribution of the mutant TPO is comparable to that seen in a normal control (Fig. 3CGo), indicating that membrane localization is probably not affected. Aside from multicentric focal medullary thyroid carcinoma associated with C-cell hyperplasia (not shown), the microscopic morphology of the remaining affected thyroid tissue is normal (Fig. 3AGo).

In silico study of the human TPO

Gln660 is located within a highly conserved region across species, which suggests an important role in the function and/or structure of TPO (Fig. 2BGo). To approach the three-dimensional structure of the Gln660Glu TPO, 1) we modeled the wild-type and mutant TPO in silico, 2) we performed a molecular dynamics simulation over 5 nsec of the wild-type and mutant TPO, and 3) we used these models to compute the effect of the Gln660Glu amino acid substitution on the protein-heme electrostatic interaction. Because the modeled site of TPO shares a high homology with MPO (50% homology overall, up to 75% for the catalytic site), the production of a reliable model is expected (31). Indeed, our model shows a good quality when assessed with the ProQ algorithm and is very similar to previously published models of the MPO-like domain (14, 16, 32). The wild-type and mutant TPO molecular dynamics simulation showed a stable structure with no amino acid clashes and the root mean square fluctuations (RMSF, a proxy of the protein backbone movement during simulation) over the 4- to 5-nsec period was very similar between mutant and wild-type TPO, with a significant (P < 0.001; n = 607) two-tailed Spearman’s correlation coefficient of 0.79 (Fig. 4Go). Together, these findings suggest that the Gln660Glu three-dimensional structure is stable and is similar to the wild-type.


Figure 4
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  		FIG. 4. The RMSF as a function of the amino acid number of the TPO protein. The RMSF was calculated for the backbone atoms (i.e. C{alpha} of amino acids) of the wild-type TPO (black line) and Gln660Glu TPO (red line) over 1 nsec of molecular dynamics simulation. Spearman’s test gives a correlation coefficient rs of 0.79 (P < 0.001) between wild-type and mutant RMSF values (n = 607).

 
We next addressed the issue of whether the Gln660Glu mutation has a major electrostatic influence on the heme group. Indeed, the substitution of a glutamine by a glutamate at position 660 changes the electrostatic potential of this residue (Fig. 5Go). Thus, even though the Gln660Glu substitution is not directly in contact with the heme, the relative electrostatic binding energy of the heme-mutant TPO interface becomes repulsive (+150 kJ/mol), whereas the relative binding energies of alternative substitutions (Gln660Ala and Gln660Gly) remained at +23 kJ/mol (Table 1Go). In other words, the heme-Gln660Glu TPO electrostatic binding energy is six times more repulsive than the concurrent value for the alternative substitutions. This repulsive trend remained independent of the conditions used for the calculation (i.e. ionic strengths and coefficient models used).


Figure 5
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  		FIG. 5. The electrostatic surface potential of the amino acid 660 of TPO: A, Gln660 in the wild-type TPO; B, the Glu660 in the mutant TPO. Both proteins are presented with the same orientation. The electrostatic surface potential for the positive (transparent blue) and negative (transparent red) isocontours are drawn at the distance where electrostatic potential is equal, respectively, to +1 or –1 kBT/e.

 

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  		TABLE 1. Relative electrostatic binding energy

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study, we describe a pedigree presenting with pseudodominant goitrous CH, and we report two novel TPO mutations. Combining immunohistochemistry and in silico modeling, we show that the Gln660Glu TPO, the most common TPO mutation in the Brazilian and Portuguese series (7, 29), is very likely to be expressed properly but has an impaired function secondary to changes in the electrostatic binding energy of the heme-TPO interface. This might impair the function of the heme within the mutant TPO.

The TPO enzyme activity depends on 1) proper folding and membrane insertion, and 2) an intact catalytic site (heme-binding region encoded by exons 8, 9, and 10) (13). Consequently, three different mechanisms can lead to decreased TPO activity. First, a frameshift mutation leads to a truncated protein, and the expression at the apical membrane is impaired due to the lack of transmembrane and intracellular domains. Second, an amino acid substitution either induces a major three-dimensional change or disrupts the glycosylation consensus sequence leading to impaired folding. Thus, the misfolded mutant TPO being trapped in the endoplasmic reticulum, its expression at the apical membrane is impaired (33). Third, an amino acid substitution might impair the function without affecting the overall structure and the localization of TPO (34). Of note, using the known structure of MPO as a model, it has been suggested that TPO is a homodimer in vivo (35). However, 1) TPO-soluble monomers are bioactive in vitro (36), and 2) the very similar MPO is also active as a monomer (37). Therefore, TPO mutants potentially impairing homodimerization are likely to have little impact, if any, on TPO enzymatic activity.

Herein, we present two different mechanisms that could explain the impaired function of the TPO in the reported family. First, we describe two new TPO mutations (c.1496delC and c.1955insT) leading to premature stop codons. The resulting truncated TPO proteins lack the entire transmembrane and intracellular part and therefore cannot be properly inserted in the apical membrane. Second, the immunochemistry results on the thyroid of the uncle (II-1), who is compound heterozygous for Gln660Glu and for a frameshift mutation, suggest that Gln660Glu TPO is properly expressed but is not functional. Indeed, the staining observed at the apical membrane accounts exclusively for the expression of the Gln660Glu mutant (Fig. 3Go, A and B), because the frameshift mutant is not recognized by the MoAB47 antibody (Fig. 2AGo). Consistent with a preserved expression on the apical membrane, our computational simulation showed no changes in the three-dimensional structure of the Gln660Glu TPO (Fig. 4Go). However, a change from a basic (glutamine) to an acidic (glutamic acid) amino acid is likely to change the electrostatic environment in the vicinity (10 Å) of the heme group (Fig. 5Go). Indeed, the computation of the heme-TPO electrostatic forces with the adaptive Poisson-Boltzman solver (26) reveals a positive relative binding energy of 150 kJ/mol (i.e. repulsive) for the Gln660Glu when compared with the wild-type TPO (Table 1Go). In other words, Gln660Glu TPO has a severely altered electrostatic environment around its catalytic site. This could explain why the function is abolished and why the patient presented with a severe phenotype, even though Gln660Glu TPO is expressed properly. Moreover, these electrostatic changes have also consequences on the redox potential of the Gln660Glu TPO, which might impair proper iodide oxidation. Nonetheless, more studies are necessary to clarify more precisely the effect of electrostatic changes on the heme stability and redox potential of TPO.

In summary, we report the first pedigree presenting with pseudodominant goitrous CH due to compound heterozygous TPO mutations. Although goitrous CH generally follows a recessive mode of inheritance, the estimated frequency of TPO mutations carriers (up to 1 in 80) may lead to pseudodominant inheritance. Furthermore, combining thyroid immunohistochemistry, patients’ phenotype, and computational modeling, we conclude that the Gln660Glu mutant TPO is very likely to be properly expressed but lacks enzymatic activity.


   Acknowledgments
 
We thank the family members described herein for their cooperation and Dr. Gilles Gariépy (Department of Pathology, University of Montreal) for the retrieval of pathological specimens.


   Footnotes
 
This work was supported by the Swiss Foundation of Medical-Biological Scholarships (PASMA-112979), by the 2007 Hoffmann-La Roche/Canadian Pediatric Endocrinology Group Fellowship, by a research grant from the Endocrine Fellows Foundation (USA), and by a fellowship grant from the Departments of Pediatrics of the University of Montreal (to J.D.); by generous donations from Mr. John H. McCall MacBain (to G.V.V.); by the University of Mainz-MAIFOR (to J.P. and N.P.); and by the Belgian Fonds de la Recherche Scientique Médicale, Fonds ERASME and Action de Recherche Concertée de la Communauté française de Belgique (to J.P. and G.V.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online November 20, 2007

Abbreviations: CH, Congenital primary hypothyroidism; MPO, myeloperoxidase; RMSF, root mean square fluctuation; TPO, thyroid peroxidase.

Received October 10, 2007.

Accepted November 13, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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