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A Novel Missense Mutation in Human TTF-2 (FKHL15) Gene Associated with Congenital Hypothyroidism But Not Athyreosis

. Bar¹, A. E. Arsoy¹, A. Smith¹, M. Agostini, C. S. Mitchell, S. M. Park, A. M. Halefolu, E. Zengin, V. K. Chatterjee and E. Battalolu

Department of Molecular Biology and Genetics (I.B., E.B.), Boaziçi University, 34342 Istanbul, Turkey; Faculty of Medicine (A.E.A., E.Z.), Department of Pediatrics, Kocaeli University, 41380 Kocaeli, Turkey; Department of Medicine (A.S., M.A., C.S.M., S.M.P., V.K.C.), Addenbrooke’s Hospital, University of Cambridge, CB2 2QR Cambridge, United Kingdom; and Department of Radiology (A.M.H.), isli Etfal Training and Research Hospital, 80220 Istanbul, Turkey

Address all correspondence and requests for reprints to: Esra Battalolu, Ph.D., Boaziçi University, Department of Molecular Biology and Genetics, Bebek, 34342 Istanbul, Turkey. E-mail: battalog{at}boun.edu.tr.

	Abstract

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Background: Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism (CH), and its genetic basis is largely unknown. Hitherto, two mutations in the human thyroid transcription factor 2 (TTF-2) gene have been described in unrelated cases of CH with cleft palate, spiky hair, variable choanal atresia, and complete thyroid agenesis. Here, we describe a novel TTF-2 mutation in a female child resulting in syndromic CH in the absence of thyroid agenesis.

Results: The index case is homozygous for an arginine to cysteine mutation (R102C) of a highly conserved residue within the forkhead, DNA binding domain of TTF-2. Her consanguineous, heterozygous parents are unaffected, and the mutation was not detected in 100 control chromosomes. Consonant with its location, the R102C mutant TTF-2 protein showed loss of DNA binding and was transcriptionally inactive. CH in the proposita was associated with cleft palate, spiky hair, and bilateral choanal atresia. However, radiological studies showed the presence of thyroid tissue in a eutopic location.

Conclusion: Our findings indicate that human thyroid development can occur despite loss of TTF-2 function and suggest that TTF-2 gene defects should also be considered in cases of syndromic CH without total athyreosis.

	Introduction

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CONGENITAL HYPOTHYROIDISM (CH), the most common neonatal endocrine disorder affecting one in 3000–4000 infants, is caused by either defects in hormone synthesis (dyshormonogenesis) or, most frequently (85% of cases), defective thyroid development (dysgenesis) (1). Whereas dyshormonogenetic causes of CH are associated with either goiter or a normal-sized thyroid gland, the spectrum of dysgenetic disorders includes thyroid ectopy, hypoplasia, and agenesis (2). Data from knockout mice have demonstrated the role of several genes in thyroid organogenesis, and accordingly, mutations in PAX8, TSH receptor, and thyroid transcription factor genes, TTF-1 (NKX2–1) and TTF-2, have been reported in rare CH cases (3).

Thyroid transcription factor 2 (TTF-2) is a member of the forkhead/winged helix-domain protein family, many of which are key regulators of embryonic development (4). The human TTF-2 gene (also known as FKHL15 or FOXE1) is expressed continuously in the thyroid from early development and consists of a single exon encoding a 42-kDa protein of 367 amino acids. TTF-2 regulates the transcription of target genes such as thyroglobulin and thyroid peroxidase (TPO) by binding to specific regulatory DNA sequences within their promoters via its forkhead DNA binding domain (5, 6, 7).

Homozygous TTF-2 null mice exhibit cleft palate and thyroid malformation consisting of either thyroid agenesis or ectopic thyroid development (8). Homozygous, human TTF-2 mutations result in a syndromic form of dysgenetic CH (Bamforth-Lazarus syndrome), whose phenotype includes thyroid agenesis, cleft palate, and spiky hair, with or without choanal atresia and bifid epiglottis, depending on the severity of the mutation (9, 10, 11). Here we describe a child, born to consanguineous parents of Turkish origin, presenting with CH, bilateral choanal atresia, cleft palate, and spiky hair. The patient was homozygous for a novel missense mutation (R102C) within the forkhead domain of the TTF-2 protein. Unlike the TTF-2 mutations described previously, radiological examination of this patient revealed the presence of thyroid tissue in an eutopic location.

	Subjects and Methods

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Mutation screening

With informed consent, genomic DNA was isolated from whole blood and the entire coding exon of TTF-2 (accession no. NM_004473) was sequenced from the index case and first-degree relatives using previously described methods (11). The creation of a new AlwNI restriction site by the mutation allowed screening of 100 control chromosomes using AlwNI (MBI Fermentas) digestion (4 h at 37 C).

Radiological studies

The presence of eutopic thyroid tissue was evaluated using 7.5 MHz ultrasonography (at birth and 4 yr) and gadolinium-enhanced computed tomography (CT) examination at age 9 yr.

Functional studies

DNA binding of wild-type and mutant TTF-2 was assessed by EMSA. Wild-type TTF-2 cDNA was amplified by PCR using genomic DNA from a normal control and cloned as a BamHI-XbaI fragment into the pcDNA3 eukaryotic expression vector (Invitrogen, Carlsbad, CA). R102C mutant TTF-2 was generated by site-directed mutagenesis of the wild-type TTF-2 template using a standard protocol (Quickchange; Stratagene, La Jolla, CA). All constructs were verified by direct sequencing. Equal amounts of ³⁵S-methionine-labeled wild-type or mutant TTF-2 proteins were generated by in vitro translation (TNT; Promega, Madison, WI), verified by SDS-PAGE analysis, and incubated for 30 min at room temperature with a ³²P-labeled oligonucleotide duplex corresponding to the TTF-2 binding site in the thyroglobulin promoter (5'-GAGGGAGTTCCTGTGACTAGCAGAGAAAAC-AAAGTGAGCCAC-3' (12), in buffer (20 mM HEPES, 10% glycerol, 2 mM dithiothreitol, 150 mM KCl, pH 7.8) in the presence of 1 µg poly (dI-dC). Protein-DNA complexes were resolved on a 6% polyacrylamide gel.

The transcriptional properties of wild-type and mutant TTF-2 were tested by transient cotransfection in 293 EBNA cells (ECACC no. 85120602) grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin, streptomycin, and fungizone (GIBCO-BRL, Carlsbad, CA). A luciferase reporter gene (hTPO-LUC) was constructed by cloning a 416-bp fragment (–366 to +50) of the human TPO promoter (13) into pA3LUC (14). Twenty-four-well plates were transfected with 500 ng reporter gene, 10 ng TTF-1 expression vector, 100 ng Bosßgal, and 1–10 ng TTF-2 expression vector using the calcium phosphate method. After 36–48 h, cells were harvested, and luciferase and ß-galactosidase assays were performed with normalization of luciferase values to ß-galactosidase activity, as described previously (14).

	Results

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Clinical features

The proband, a 3900-g female infant, was born to consanguineous parents by normal vaginal delivery at 40 wk gestation after an uncomplicated pregnancy. Postnatal examination revealed the patient to be hypotonic, hypoactive, hypothermic, and areflexic with cleft palate, spiky hairs, and bilateral choanal atresia, subsequently confirmed by paranasal sinus tomography. Meconium staining and perinatal respiratory distress prompted admission to neonatal intensive care for ventilatory support. Immediately after birth, the patient’s total serum T₄ level was 0.758 µg/dl [normal range (NR), 6.1–14.9 µg/dl], and TSH was greater than 100 mIU/ml (NR, 1.7–9.1 mIU/ml). L-T₄ replacement therapy was started, and the baby was discharged at age 2 months.

Follow-up surveillance at age 19 months showed no neurological abnormalities, and thyroid function tests on L-T₄ therapy revealed TSH 0.57 mIU/ml, total T₃ (TT₃) 1.64 ng/ml (NR, 0.8–2.0 ng/ml), total T₄ (TT₄) 11.3 µg/dl, free T₃ (fT₃) 3.94 pg/ml (NR, 2–4.4 pg/ml), free T₄ (fT₄) 1.85 ng/dl (NR, 0.9–1.7 ng/dl), and thyroglobulin less than 0.2 ng/ml (age-related NR, 1–110 ng/ml). Reassessment at age 9 yr, after withdrawal of L-T₄ for 7 wk, showed profound biochemical hypothyroidism (TT₄, <1 µg/dl; TT₃, <0.4 ng/ml; TSH, 357 mIU/ml; fT₃, <1 pg/ml; and fT₄, <0.3 ng/dl). ¹³¹I scanning showed negligible uptake, and serum thyroglobulin was 0.1 ng/ml (age-related NR, 2–65 ng/ml). The proband’s mother (TT₄, 5.82 µg/dl; TSH, 2.33 mIU/ml), father (TT₄, 9.3 µg/dl; TSH, 1.42 mIU/ml) and older male sibling (TT₄, 7.26 µg/dl; TSH, 3.61 mIU/ml) are biochemically euthyroid with no congenital anomalies.

Radiological investigations

Neck ultrasonography performed at birth and age 4 revealed heterogeneous, hyperechoic tissue in the paratracheal region corresponding to a eutopic thyroid location. Recent CT examination confirmed solid tissue in this location (Fig. 1A) consisting of right and left lobes and isthmus [anterior-posterior diameters 1.96, 2.02, and 0.48 cm, respectively (normal diameter, <1.5 cm)], which failed to enhance with contrast, unlike normal, functioning thyroid tissue (Fig. 1B).

View larger version (68K): [in this window] [in a new window]   FIG. 1. A, Axial postcontrast CT image of the patient reveals a slightly enlarged thyroid gland (arrows) in the paratracheal region with absent contrast enhancement. B, Comparative imaging of the thyroid gland (arrows) in a healthy 9-yr-old child. Alignment of the forkhead, DNA-binding domain of TTF-2 with this region from selected FOX proteins in different species (C) and in different human FOX proteins (D). *, Conserved residues in all sequences; , arginine 102 residue.

Direct sequencing of the coding exon of TTF-2 gene revealed the proband to be homozygous for a single nucleotide substitution (CGC to TGC) at position 304, corresponding to an arginine to cysteine mutation at codon 102 (R102C) within the forkhead, DNA binding domain of the predicted protein sequence. Both parents were heterozygous for the mutation, consistent with an autosomal recessive mode of inheritance. AlwN1 digest confirmed that the R102C mutation was absent in 100 control chromosomes tested.

Functional characterization

Consonant with the location of the R102C substitution within the forkhead, DNA binding domain of TTF-2 and the conservation of this residue in FOX proteins from different species or humans (Fig. 1, C and D), EMSAs showed negligible binding of the R102C mutant TTF-2 protein to a known TTF-2 response element (Fig. 2B).

View larger version (17K): [in this window] [in a new window]   FIG. 2. Functional properties of the TTF-2 mutant. A, Transcriptional activity of wild-type (WT) and mutant (R102C) TTF-2 on the human TPO promoter. 293 cells were transfected with hTPO-LUC and 10 ng TTF-1 expression vector, together with increasing amounts (1–10 ng) of empty pcDNA3, wild-type (WT), or mutant (R102C) TTF-2 expression vectors. Fold induction of reporter gene activity by coexpressed TTF-2, relative to empty vector transfected cells, is shown, and the results represent the mean (SEM) of three independent transfections each performed in triplicate. B, DNA binding of wild-type (WT) and mutant (R102C) TTF-2. 35S-labeled in vitro translated WT and R102C TTF-2 proteins were bound to a 32P-radiolabeled oligonucleotide probe corresponding to the TTF-2 binding site in the thyroglobulin promoter. WT forms a specific complex (arrowhead), whereas the R102C protein fails to form this complex, although the inset (C) shows comparable synthesis of each protein when analyzed by SDS-PAGE. RL, Unprogrammed reticulocyte lysate.

The complete lack of DNA binding and transcriptional activity of the R102C mutant are more analogous to the properties of the A65V TTF-2 mutant described previously (10) than the partial preservation of function noted with the S57N mutant (11).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We describe a child with CH associated with bilateral choanal atresia, cleft palate, and spiky hair who is homozygous for a novel, missense mutation (R102C) within the forkhead DNA binding domain of TTF-2. Both parents were heterozygous for the mutation but euthyroid with no congenital anomalies, and the mutation was absent in 100 control chromosomes. The arginine residue at codon 102 is a highly conserved residue in the forkhead domain family of proteins (Fig. 1, C and D), and mutations of the equivalent amino acid in other forkhead proteins [e.g. FOXC1 (R127H), FOXC2 (R121H), and FOXP2 (R55³H)] have been described. Consonant with this, in vitro studies indicate that the mutation is highly deleterious, with the R102C mutant protein exhibiting negligible DNA binding and transcriptional activity. Interestingly, thyroid ultrasonography and CT examination of the proband indicated thyroid tissue in a eutopic location, although biochemical measurements and radioisotope scanning show that it is nonfunctional.

Our case represents the third recorded example of a loss-of-function mutation in the human TTF-2 gene, with the two previously described mutations (A65V and S57N) also being located within its forkhead, DNA-binding domain (10, 11). The A65V mutation was identified in a nonconsanguineous Welsh family with two male siblings exhibiting CH, cleft palate, choanal atresia, and bifid epiglottis together with spiky hair, whereas the S57N mutation was reported in two male siblings of a consanguineous Tunisian kindred exhibiting CH and cleft palate alone. With both of these TTF-2 mutation cases, ¹²³I scanning and ultrasonography showed complete athyreosis (10, 11). In contrast, although the third case we report here shares some of these features, including severe CH and extrathyroidal anomalies, imaging indicates the presence of thyroid tissue in a eutopic location. However, severe biochemical hypothyroidism at birth and also following T₄ withdrawal, together with absent ¹³¹I uptake and very low serum thyroglobulin levels, indicates that the function of such glandular tissue is markedly compromised.

Mouse models support a critical role for TTF-2 in thyroid and palate organogenesis. Expression of TTF-2, together with the transcription factors TTF-1 and PAX-8, has been demonstrated from the onset of formation of the thyroid primordium (embryonic d 8–8.5), continuing throughout the migration of the thyroid diverticulum (4, 16, 17). TTF-2 is also expressed in craniopharyngeal ectoderm involved in palate formation and Rathke’s pouch in mouse embryos (4) and in the outer follicular hair sheath in humans (18). Targeted disruption of the murine Titf2 locus results in homozygous null mice with cleft palate and either complete thyroid agenesis or ectopic sublingual gland development (8). The two murine phenotypes were seen with equal frequency and may reflect different developmental manifestations of the disorder. Thus, similar to the murine context, our human proband illustrates that thyroid morphogenesis can occur in the absence of TTF-2, albeit with migration of thyroid gland tissue to a eutopic location.

Mutations in TTF-2 and a TTF-1/NKX2.1 mutation (19) are the only known genetic causes of thyroid agenesis. However, involvement of TTF-2 accounts for only a small minority of CH cases, being a strong consideration only in those with cleft palate, which is an infrequent association of CH (20). This case illustrates further phenotypic heterogeneity associated with human TTF-2 mutations and suggests that defects in this gene should also be considered in cases of syndromic CH with cleft palate, but not necessarily complete thyroid agenesis. This variable phenotype may reflect differential effects of TTF-2 mutations on downstream target genes required for normal human thyroid organogenesis, migration, and differentiation, and the further identification of such genes may elucidate these mechanisms and provide novel genetic candidates for CH and cleft palate.

	Acknowledgments

 
We thank the participating family for their cooperation throughout the study.

	Footnotes

 
This work was partially supported by a grant from Boaziçi University Research Fund (03S101). A.S., M.A., C.S.M., and V.K.C. are supported by the Wellcome Trust.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 1, 2006

¹ I.B., A.E.A., and A.S. should be regarded as joint first authors.

Abbreviations: CH, Congenital hypothyroidism; CT, computed tomography; fT₃, free T₃; fT₄, free T₄; NR, normal range; TPO, thyroid peroxidase; TT₃, total T₃; TT₄, total T₄; TTF-2, thyroid transcription factor 2.

Received February 21, 2006.

Accepted July 26, 2006.

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