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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-1497
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 5 1832-1841
Copyright © 2006 by The Endocrine Society

Functional Study of a Novel Single Deletion in the TITF1/NKX2.1 Homeobox Gene That Produces Congenital Hypothyroidism and Benign Chorea But Not Pulmonary Distress

Christian M. Moya1, Guiomar Perez de Nanclares1, Luis Castaño, Neus Potau, J. Ramón Bilbao, Antonio Carrascosa, María Bargadá, Raquel Coya, Pedro Martul, Enric Vicens-Calvet and Pilar Santisteban

Biomedical Research Institute Alberto Sols (C.M.M., P.S.), Spanish National Research Council-Autonomous University of Madrid, E-28029 Madrid, Spain; Endocrinology and Diabetes Research Group (G.P.d.N., L.C., J.R.B., R.C., P.M.), Hospital de Cruces, University of Basque Country, Barakaldo, E-48902 Basque Country, Spain; and Hormone Laboratory (N.P.) and Departments of Endocrinology (A.C., E.V.-C.) and Psychology (M.B.), Children’s Hospital Vall d’Hebron, Autonomous University of Barcelona, E-08035 Barcelona, Spain

Address all correspondence and requests for reprints to: Pilar Santisteban, Biomedical Research Institute Alberto Sols, Spanish National Research Council-Autonomous University of Madrid, Madrid, Spain. E-mail: psantisteban{at}iib.uam.es; or evicens{at}vodafone.es.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: We studied two sisters with congenital hypothyroidism and choreoathetosis but not respiratory distress.

Objective: The aim of this study was to establish the genetic defect that causes this phenotype and study the molecular mechanisms of the pathology by means of functional analysis.

Design: Sequencing of DNA, expression vectors generation, EMSAs, transfections experiments as well as bioinformatics analysis were performed.

Results: We found a new single deletion (825delC) in one allele of the TITF1/NKX2.1 gene. The mutation located in the C-terminal domain generates a nonsense thyroid transcription factor 1 (TTF1) protein, with 22 amino less and rich in positive charges. This protein shows diminished binding to DNA, does not interfere with wild-type (wt) TTF1 binding, and fails to activate reporter genes harboring the thyroglobulin (Tg), thyroperoxidase (TPO), or surfactant protein B (SP-B) promoters. In addition, the mutant (mut) protein has a dominant-negative effect on the transcriptional activity of wt TTF1 in a promoter-specific manner, inhibiting the transcription of Tg and TPO but not of SP-B. Using a Gal4 reporter system, we demonstrate that the mut protein is not transcriptionally active and does not likely compete with the wild type for coactivators. Interestingly, the mut protein impairs the wt capacity to synergize with paired box 8 (PAX8). This cooperation is necessary for Tg and TPO transcription but dispensable for SP-B expression.

Conclusion: These results are concordant with the phenotype of the two sisters studied and demonstrate a differential role for TTF1 in the different tissues in which it is expressed.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
NEUROLOGICAL SYMPTOMS AND maturation delay in newborns with congenital hypothyroidism (CH) have been attributed to thyroid hormone deficiency in the developing brain. Neonatal screening programs for CH, early diagnosis, and appropriate treatment permit normal progression for most CH patients, whereas unfavorable outcome is considered to be due to late diagnosis and/or inadequate treatment. In rare cases, however, neurological outcome is poor, even after adequate hormone replacement; in these patients, retardation of psychomotor development may be related to other defects, such as an underlying genetic condition. Mutations in the paired box 8 (PAX8) (1, 2, 3, 4, 5) and forkhead FOXE1 [formerly called TTF2 (thyroid transcription factor 2)] (6, 7) genes have been associated with thyroid dysgenesis but never neurological diseases. Recently several cases of neurological problems associated with thyroid abnormalities that are due to mutations in the thyroid transcription factor 1 (TITF1/NKX2.1) (8, 9, 10, 11, 12, 13, 14) and the monocarboxylate transporter (MCT8) (15, 16) have been described.

TITF1/NKX2.1 [formerly termed TTF1 (thyroid transcription factor 1) or thyroid/enhancer binding protein] is a member of the NK-2 gene family of transcription factors (17), which was first identified as a nuclear protein able to bind a specific sequence in the thyroglobulin (Tg) gene promoter (18, 19). TITF1/NKX2.1 expression is not restricted to the thyroid because it has also been observed in forebrain and lung during embryogenesis (20, 21). Developmental defects of these structures were described in null TITF1/NKX2.1 mice characterized by thyroid agenesis, transformation of hypothalamus pallidum to striatum, and lung hypoplasia with severe respiratory failure, which is responsible for lethality at birth (22, 23). Based on the thyroid dysgenesis phenotype of the null mice, human TITF1/NKX2.1 was postulated as a candidate gene for CH, although no mutations have been found so far in patients with a phenotype restricted to thyroid dysgenesis (24, 25, 26). In contrast, several patients with chromosomal deletions (8, 9, 11, 12) and point mutations in this gene (10, 11, 12, 13, 14) had a complex phenotype that included thyroid, respiratory, and neurological defects, with benign hereditary chorea (BHC) being the most frequent among the neurological problems. This autosomal dominant disease is defined by rapid involuntary and slow writhing movements due to defects in the basal ganglia, a brain region in which TTF1 is expressed during development. Moreover, the BHC is linked to a region on chromosome 14 that includes the TITF1/NKX2.1 gene (27) and is normally associated with mild or severe hypothyroidism and, in most cases, respiratory problems.

Here we report two sisters, detected in a screening for CH, that present mild hypothyroidism in addition to choreoathetosis but no respiratory distress. We found a deletion of cytosine 825 (825delC) in exon 3 in one allele of TITF1/NKX2.1 gene; functional studies showed that the mutated nonsense protein can act in a dominant negative fashion, impairing the transcriptional activity of the wild-type (wt) TTF1 by inhibiting its cooperation with PAX8.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

Case 1. She had a birth weight of 4300 g, birth length of 52 cm, and Apgar score 7/10. The neonatal screening data determined at postnatal d 18 were TSH of 186.2 mU/liter (upper reference value, 16.9 mU/liter) and T4 of 6.5 µg/dl (lower reference value, 9.6 µg/dl). Thyroid scintigraphy revealed a normotopic, bilobulated gland with low uptake. Transient hypothyroidism was initially suspected. Treatment was finally begun based on the high levels of TSH and low-normal T4 values, which normalized with low doses of levothyroxine (6–8 µg/d). Doses increased with age and at the age of 11 yr, 100 µg/d were required. At 15 months, the child did not walk and choreoathetotic movements that hindered psychomotor progression appeared. Slight psychomotor delay also became evident at that age.

Case 2. A sister was born 6 yr later (birth weight, 4330 g; birth length, 50 cm) with similar biochemical characteristics. The neonatal screening data determined at postnatal d 17 were TSH of 65.0 mU/liter (upper reference value, 14.3 mU/liter) and T4 of 9.7 µg/dl (lower reference value, 21.2 µg/dl). Treatment was begun at 2 months of age, when TSH values remained high and T4 values declined. Scintigraphy and follow-up, with the appearance of choreoathetotic movements, mirrored her sister’s history. No pulmonary involvement was observed in either case. Brain magnetic resonance imaging performed at age 7 yr in case 1 and at age 4 yr in case 2 was normal.

In view of this second case, a detailed family history was taken, which revealed that the mother had had transient hypothyroidism during both pregnancies and that the maternal grandmother had suffered a mild form of chorea with no lifelong mental impairment, but no records of hypothyroidism were found. The mother currently has TSH levels of 10–12 mU/liter and normal T4 and free T4 values and bears the same deletion as her daughters. The choreoathetosis history of the grandmother was reported by the family, but no mutational study was performed (Fig. 1Go). In all cases, study protocols were approved by the Vall d’Hebron Hospital Institutional Review Board committee.


Figure 1
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  		FIG. 1. Family pedigree showing the autosomal dominant inheritance pattern with variable clinical expression of the mut TITF1/NKX2.1 allele in carriers. The mother and her daughters are heterozygous carriers of the 825delC mutation; although the grandmother is dead, the symptoms described by the family suggest that she also carried the mutation. Black symbols represent hypothyroidism with BHC. The half-black symbol (left) represents BHC and (right) mild hypothyroidism. Open symbols indicate unaffected individuals. A question mark denotes uncertain diagnosis. Arrows indicate the index patients.

 
Psychomotor evaluation

Tests used were the Denver developmental test (18 months of age, case 2); the McCarthy cognitive test, Luria’s neuropsychological test and projective emotional tests (4 yr of age, cases 1 and 2), and the Wechsler Intelligence Scale for Children-Revised test (5, 8, and 11 yr, case 1).

Mutational analysis

Genetic analyses were performed after informed consent of all individuals studied and approved by the institutional review board committee. Genomic DNA of the subjects was extracted from peripheral blood leukocytes according to the manufacturer’s instructions (NucleoSpin Blood; CLONTECH, Madrid, Spain). The three coding exons of the TITF1/NKX2.1 gene were amplified using sequences and conditions kindly provided by Dr. Samuel Refetoff (University of Chicago, Chicago, IL), sequenced in both directions (sense and antisense) and loaded onto an ABI PRISM 3100 Avant DNA sequencer (Applied Biosystems, Madrid, Spain).

Plasmids

Plasmids used in this work were human cDNA wt TTF1 and Pax8 cloned in pcDNA3 (10, 28) and reporter vectors, hTGenh/prom-Luc and hTPOprom-Luc cloned in pGL3 Basic (5, 10) and hSP-Bprom-Luc (B-500) cloned in pGL2 Basic (29); pG5-luciferase reporter plasmid has five copies of Gal4 DNA binding sites (30), and pM2 vector contains the DNA-binding domain of Gal4.

Site-directed mutagenesis

The human mut TTF1 (825delC) was generated by site-directed mutagenesis using asymmetric PCR and a single mutated primer (31, 32). The method involves two steps: the first PCR uses the mutant (mut) oligonucleotide containing the described mutation (reverse primer mut: 5'-GCG CCC GGC GCG GGG CAC CCG CC-3'; the wt sequence has five G’s in the underlined region) and a nearby flanking primer (forward primer: 5'-GGG GGC GGC GGG GGC ACC GGG-3'). The resulting product (127 bp) together with reverse primer II (5'-GAG GGC GGT CGC CGC TGA GCC-3') was then used in a second round of PCR. The amplifications were performed with 5–50 fmol of plasmid (pcDNA3 wt TTF1) in 50 µl containing 10x buffer Pfu Turbo, 10 mM deoxynucleotide triphosphate, 10 pmol 5'-flanking primer, 10 pmol antisense mut primer (in second PCR, 20 pmol amplified mut fragment were used), and 1 U Pfu Turbo polymerase (Stratagene, Madrid, Spain). Amplification was done with an annealing temperature of 68 C for 40 cycles and 10 min of final extension. Products of the second PCR (990 bp) and pcDNA3 wt TTF1 were digested with BlpI. After each step, PCR products and vectors were purified by gel electrophoresis and the GFX PCR DNA and gel band purification kit (Amersham Biosciences, Barcelona, Spain), according to the manufacturer’s protocols. The pcDNA3 fragment was dephosphorylated, and both DNAs were ligated to generate pcDNA3 mut TTF1. The vector with the deletion (825delC) was sequenced to confirm the presence of the mutation (Fig. 2CGo).


Figure 2
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  		FIG. 2. Identification of the mutation in the TITF1/NKX2.1 gene and amino acid sequence of the wt and mut TTF1 proteins. Fragments of sequence chromatograms from an unaffected individual (A), a patient (B), and the vector construct harboring the mutation (C). The arrow indicates deletion of a cytosine at position 825 of the TITF1/NKX2.1 cDNA (position 1 is the first coding nucleotide in exon 2). The heterozygous mutation produces a double sequence beyond the deletion site. The encoded amino acids in wt and mut TTF1 are indicated. D, Amino acid sequence comparison of wt and mut (bold) TTF1 proteins, deduced by bioinformatics programs. The homeobox (gray box), glutamine/alanine-rich regions (blue), cysteines for dimerization (green), and serines involved in phosphorylation (red) are shown. The positively charged amino acids in the nonsense region of mut TTF1 are in a black box and are indicated with +. Important amino acids absent from the mutated protein are indicated in the wt protein with the colors blue, green, and red having the same meaning as indicated above.

 
Generation of Gal4 constructs

The Gal4-wt TTF1 and Gal4-mut TTF1 chimeras were constructed in the plasmid pM2. The TTF1 wt and mut cDNAs were obtained by PCR from the expression vectors generated above using two primers with specific restriction sites to allow subcloning into the BamHI and HindIII sites of pM2 (forward primer: 5'-cgc cgg aat tcc gga tcc TCG ATG AGT CCA AAG CAC AC-3'; reverse primer: 5'-ac tta tct aga caa gct tTC TCA CCA GGT CCG ACC GTA T-3', TTF1 sequence are in capital letters). All steps were performed under the same conditions as the PCR for site-directed mutagenesis. Sequencing of the generated constructs confirmed that they correspond to the wt and mut TTF1 cDNAs, respectively.

In vitro synthesis of TTF1 and EMSA

Proteins were synthesized from wt and mut TTF1 vectors by in vitro transcription/translation using the TNT coupled reticulocyte lysate system (Promega, Madrid, Spain). For EMSAs, the in vitro-translated proteins were mixed with 32P-labeled oligonucleotide C (33), C' (34), or surfactant protein B (SP-B)-f1 (35) derived from the Tg, thyroperoxidase (TPO), and SP-B promoters, respectively, were performed as previously described (33). In supershift experiments, an excess of related anti-TTF1 (BioPat, Piedimonte Matese, Italy) or unrelated anti-specificity protein-1 (Sp1) (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were added before probe addition.

In vitro transactivation assay

HeLa cells were transfected as previously described (33) with 3 µg reporter plasmid DNA per dish and different amounts of wt, mut, and empty/carrier vector, as indicated in each experiment. To correct for transfection efficiency, 50 ng of Renilla-encoding pRL-Tk vector was added in all cases. After 48 h, cells were harvested, lysed, and analyzed for luciferase (LUC) and Renilla activity using the dual-LUC reporter assay system (Promega). The ratio between the LUC and Renilla activities was expressed relative to the ratio obtained in cells transfected with reporter and empty expression vector (pcDNA3) only.

Analysis of TTF1 protein

Aliquots of in vitro-transcribed/translated protein or 25 µg of total extracts from transfected HeLa cells, quantified according to Bradford (Bio-Rad Laboratories, Madrid, Spain), were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Ponceau S staining showed equal protein loading. Membranes were blocked and incubated with anti-TTF1 antibody (BioPat). Immunoreactive bands were visualized with Luminol Western blot detection reagent (Santa Cruz).

Bioinformatic analysis

Results were analyzed using the DNASTAR (DNASTAR, Inc., University of California, San Francisco, CA), nucleotide BLAST (http://www.ncbi.nlm.nih.gov/BLAST), and ExPASy proteomics server (http://www.expasy.org/) tools and software programs. Prediction of secondary structure was performed by SOPMA (36) (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html).

Statistical analysis

Statistical comparisons were done using Student’s paired t test, and the significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Psychomotor evaluation

Case 1. In the first evaluation at the age of 5 yr, notably poor motor skills were observed, particularly in balance and gross motor scale. Comparative study of the Wechsler Intelligence Scale for Children-Revised tests at ages 5, 8, and 11 yr revealed a slight decline in verbal scale and an even slighter decline in manipulative skills but always within the normal range. Comparison of parameters for attention, memory, and motility in the Luria test showed slight memory and attention deficits in each evaluation.

Case 2. At the age of 4 yr, loss of balance, tremor, hyperreflexia, and mental retardation were observed in the McCarthy test, with a marked decline in verbal performance and motor skills. Both were below the normal range.

The family pedigree is shown in Fig. 1Go, and the two patients studied are indicated by an arrow.

Mutation analysis

Sequencing analysis of the three coding exons of the TITF1/NKX2.1 gene in both patients (Fig. 2BGo) revealed a heterozygous deletion of a cytosine (C) in the third exon, affecting nucleotide 825 (825delC; GenBank accession no. NM_003317). This mutation generates a frameshift and a nonsense protein that lacks the correct transactivation domain in the C-terminal region (30). The frameshift occurs at codon 275, resulting in an aberrant protein starting at codon 276 because proline 275 was unaltered. It extends 74 amino acids and finishes at a premature stop at codon 350, producing a protein that lacks 22 amino acids, compared with the wt protein (Fig. 2DGo). Genetic analysis of the family showed that the mother also carried the mutation (data not shown).

The normal and mutated TITF1/NKX2.1 gene, generated by site-directed mutagenesis (Fig. 2CGo), were expressed in TNT and analyzed by SDS-PAGE and Western blotting (Fig. 3AGo). Although the predicted molecular weight for the mutated protein is lower than for the wt, the size observed in the gel was slightly higher. We interpret this difference as being due to the generation in the mutated protein of 20 new amino acids with a strong (43%) increase in positive charge because of the lysine- and arginine-rich nonsense region, resulting in a protein with a slightly slower electrophoretic mobility than the wt form. When both wt and mut TTF1 are expressed, a weaker band of a lower molecular weight appears that corresponds to a product translated from a downstream ATG, as described elsewhere (37, 38). The protein generated is recognized in Western blots by anti-TTF1 antibody, indicating that the mutated protein conserves immunological properties of the wt. The C-terminal region of the mutated protein has lost the glutamine/alanine-rich third domain, which has been described as necessary for TTF1 transactivation (30). Two serines (S327 and S336) involved in TTF1 phosphorylation (39) and a cysteine (C362) required for TTF1 dimerization (40) are also absent (Figs. 2DGo and 3BGo). The same apparent molecular weight was observed when the protein was expressed in eukaryotic HeLa cells (data not shown).


Figure 3
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  		FIG. 3. Western blot analysis and schematic representation of TTF1. A, Plasmid DNA (1 µg) containing wt TTF1 or mut TTF1 cDNA served as a template in a coupled transcription/translation reaction in the TNT system. The in vitro translation products (2 µl) were analyzed by Western blotting. Molecular weight standards are indicated at the left and the corresponding size of wt and mut TTF1 at the right. Both proteins were recognized by an anti-TTF1 antibody. The mutated protein migrates more slowly than wt TTF1, despite having 22 amino acids less. A weaker band of a lower-molecular-weight protein represents a product translated from a downstream ATG, as described (37 38 ). B, Schematic representation of wt and mut TTF1 proteins showing the most important regions for activity, using the color code described in Fig. 2DGo. The theoretical molecular weight (MW), isoelectric point (pI), and total and charged amino acids are indicated.

 
DNA binding capacity of mutated and wt TTF1 proteins

To characterize the mechanism by which the 825delC deletion causes disease, we studied the ability of the mut to bind DNA. We used oligonucleotides derived from the TTF1 binding sites within the Tg (oligo C), TPO (oligo C'), or SP-B (oligo SPB-f1) promoters. Although the deletion is outside the DNA-binding domain, the mut protein has a greatly reduced ability to bind to its binding site in the Tg (Fig. 4AGo, lanes 7 and 8), TPO (Fig. 4BGo, lane 5), and SP-B (Fig. 4CGo, lanes 7 and 8) promoters. The mutated TTF1/DNA complex appears as a diffuse band, less retarded than wt; it is nonetheless specific because binding is abolished by an excess of the related oligonucleotide (Fig. 4AGo, lane 10; 4B, lane 6; and 4C, lane 9) but not an unrelated oligonucleotide (Fig. 4AGo, lane 9). Specificity of binding by the wt protein was confirmed in competition experiments (Fig. 4AGo, lane 6; 4B, lane 4; and 4C, lane 6). Equal amounts of mutated TTF1 protein did not interfere with wt TTF1 binding (Fig. 4AGo, lane 11; and 4C, lane 10). When we used a 2.5-fold excess of mutated protein, we observed slight interference with wt TTF1-Tg oligo C complex formation (Fig. 4AGo, lane 12) but not with wt TTF1-SPB-f1 complex formation (Fig. 4CGo, lane 11). The interference observed in a representative EMSA (Fig. 4AGo, lane 12) was calculated as a percentage of the wt retarded band intensity in four independent experiments; statistical analysis indicated that the decrease is not significant (data not shown).


Figure 4
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  		FIG. 4. DNA-binding activity of the mutated TTF1 protein. A, wt or mut TTF1 proteins translated from TNT reticulocytes were incubated alone (lanes 3–6 and 7–10, respectively) or in combination (lanes 11–14) with the C oligonucleotide derived from the Tg promoter. Empty pcDNA3 vector was used as control (lane 2). For competition, a 100-fold excess of unrelated Sp1 (lanes 5, 9, 13) or related C (lanes 6, 10, 14) oligonucleotides were used. The amount of lysate (in microliters) added to each lane is indicated at the top of the figure. B, EMSA with wt or mut TTF1 protein translated from TNT reticulocytes (lanes 3 and 4 and 5 and 6, respectively) and the oligonucleotide C' derived from the TPO promoter. pcDNA3 was used as control (lane 2). For competition, a 100-fold excess of oligo C' (lanes 4 and 6) was used. The amount of lysate (in microliters) added to each lane is indicated at the top. C, wt or mut TTF1 proteins translated from TNT reticulocytes were incubated alone (lanes 4–6 and 7–9, respectively) or in combination (lanes 10–12) with the SPB-f1 oligonucleotide derived from the SP-B promoter. Empty pcDNA3 vector was used as control (lanes 2 and 3). For competition, a 100-fold excess of related SPB-f1 (lanes 3, 6, 9, and 12) oligonucleotides were used. The amount of lysate (microliters) added to each lane is indicated at the top. Note that the band of the wt TTF1/DNA complex migrates to the same position as the nonspecific band but that the TTF1 band is competed by the specific competitor and the unspecific band is not.

 
Supershift experiments using a specific anti-TTF1 antibody showed that the protein/DNA complexes observed with oligo C or SPB-f1 probes contain the TTF1 moiety (Fig. 5Go, A and B, lanes 4, 7, and 10). Supershift specificity was demonstrated using an unrelated anti-Sp1 antibody (Fig. 5Go, A and B, lanes 5, 8, and 11). The supershifts generated by the mutated TTF1 protein were very weak (Fig. 5Go, A and B, lane 7) due to its extremely low DNA binding ability. When wt and mutated proteins were combined, the supershift obtained was equal to that observed with wt alone (Fig. 5Go, A and B, lane 10). In addition, the supershift complex was displaced by an excess of unlabeled oligonucleotide (Fig. 5Go, A and B, lane 12). Taken together, the data show that in the heterozygous patients the mut TTF1 does not interfere with the DNA binding of the wt.


Figure 5
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  		FIG. 5. The mutated TTF1/DNA complex is recognized by an anti-TTF1 antibody. wt or mut TTF1 proteins translated from TNT reticulocytes were incubated alone (lanes 3–5 and 6–8, respectively) or in combination (lanes 9–12) with the Tg C site (A) or the SPB-f1 element (B), as described in the legend to Fig. 4Go. A clear supershift was observed when an anti-TTF1 antibody was added to the binding reaction (lanes 4, 7, and 10, both panels) but not when an unrelated anti-Sp1 antibody was added (lanes 5, 8, and 11, both panels). The specificity of the shift and supershift complexes was demonstrated by competition with an excess of related oligonucleotide (lane 12, both panels).

 
Transactivation capacity of mutated and wt TTF1 proteins

Therefore, we studied the transactivation activity of the mutated protein because the mutation occurs in one of the two transactivation domains (30). The mutated protein failed to transactivate transcription from reporters harboring the human Tg enhancer/promoter (Fig. 6AGo), the human TPO promoter (Fig. 6BGo), or the human SP-B promoter (Fig. 6CGo). The mutated protein interfered with the transcriptional activity of wt TTF1 protein only when we used the Tg enhancer/promoter (Fig. 6AGo) or the TPO promoter (Fig. 6BGo), but did not interfere with transcription from the SP-B promoter, even with increased amounts of mutated protein (Fig. 6CGo). The Tg promoter was most affected because equal amounts of both proteins decreased Tg transcription to 15–20%; the dominant-negative effect was less pronounced for TPO (50–55% decrease). These data suggest that the mutated TTF1 protein acts in a dominant-negative way and a promoter-specific manner, possibly due to competition for available activators or coactivators.


Figure 6
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  		FIG. 6. Transcription activation by wt and mut TTF1 from the hTg enhancer/promoter (A), the hTPO promoter (B), and the hSP-B promoter (C). Promoter constructs were cotransfected into HeLa cells with the empty pcDNA3 expression vector or the expression vector harboring wt or mut TTF1 cDNA. The amounts and combinations used are indicated. Promoter activity is expressed as fold induction, relative to the activity observed in the presence of empty expression vector; luciferase activity is normalized to Renilla activity derived from the cotransfected pRL-Tk vector to adjust for transfection efficiency. Results are mean ± SD of four independent experiments. **, P < 0.01; *, P < 0.05. ns, Not significant. hTg, Human Tg; hTPO, human TPO.

 
To further understand the dominant-negative action and investigate whether mut TTF1 intrinsically binds coactivators, we generated a Gal4 fusion protein (41) harboring the wt or mut TTF1. We found that the wt protein is able to activate the transcription of a reporter vector containing 5x Gal4-binding domain (pG5), whereas the mut has lost this activity and represses in part the activity of the wt (Fig. 7AGo, gray bars). Thus, competition for coactivators does not seem to explain the mechanism of action of mutated TTF1. To look more closely at the dominant-negative effect of mut TTF1, a vector expressing human PAX8 was cotransfected. This transcription factor is expressed in thyroid but not in lung and synergizes with TTF1 to stimulate Tg and TPO promoter activities (42). We found that PAX8 synergized with wt TTF1, increasing Tg promoter activity but failed to increase the activity of mutated protein. Interestingly, the mutated TTF1 protein interferes with the capacity of the wt to cooperate with PAX8, decreasing the levels of activation (Fig. 7BGo, black bars). These results were reproduced in the Gal4 system, in which mutated protein diminished the TTF1/PAX8 cooperation to almost undetectable levels (Fig. 7AGo, black bars). These results explain, at least in part, the phenotype of the two patients studied in this work.


Figure 7
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  		FIG. 7. A, Transcription activation by Gal4-wt TTF1 and Gal4-mut TTF1 (gray bars) and interactions with PAX8 (black bars) from the pG5 reporter vector (5x Gal4-binding domains). B, Transcription activation by wt and mut TTF1 (gray bars) and interaction with hPAX8 (black bars) on the hTg enhancer/promoter. Promoter constructs were cotransfected into HeLa cells with the pcDNA3 or pM2 expression vectors empties or harboring wt or mut TTF1 (gray bars). The black bar represents the experiments in which the hPAX8 expression vector was included in the transfection. The amounts and combinations used are indicated. Promoter activity is expressed as fold induction, relative to the activity observed in the presence of empty expression vector as explained in the legend of Fig. 6Go. Note that wt TTF1 synergizes with PAX8, but mut TTF1 impairs this synergism and even represses the transcriptional activities of both wt TTF1 and PAX8. Results are mean ± SD of four independent experiments. **, P < 0.01. hTg, Human Tg.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Here we describe a new mutation in the TITF1/NKX2.1 gene in two sisters affected by choreoathetosis and hypothyroidism but without respiratory distress. The fact that both patients were correctly treated with levothyroxine suggests that the neurological problems are related to gene impairment in the brain rather than a consequence of hypothyroidism. Homozygous mutations of this gene in humans have not been described, probably due to the lethal effect of these abnormalities. The two sisters studied here have a heterozygous point mutation in the gene; we present functional evidence that this deletion is responsible for the phenotype described. Family analysis indicated that the mother carries the same mutation; she had mild and transient hypothyroidism during gestation, but no choreoathetotic movements were reported. If this is correct, the mother represents the first case described in which a TTF1 mutation causes only thyroid alterations. Information provided by the family indicated that the grandmother also had choreoathetotic movements during childhood that later disappeared; no symptoms of hypothyroidism were reported, perhaps because thyroid function was not studied at that time. The variable phenotype in the family may be explained by differences in modifier genes that produce a different genetic background in a same family.

Since the first report of BHC (43), a large number of families have been reported with this condition, with considerable intra- and interfamilial phenotypic variation. The locus was mapped to chromosome 14q on which the human TITF1/NKX2.1 gene is located (27). This gene is composed of three exons and produces two major transcripts (2.5 and 2.3 kb) that encode proteins of 401 and 371 amino acids, respectively (37, 38). The most active and abundant polypeptide is the shorter isoform (371 amino acids), which translates from the first ATG in the second exon (44). This isoform is encoded by the wt vector used in this study (10), and the 825delC mutation is based on this shorter transcript.

Mutations in this gene were recently described in patients with CH having neurological and pulmonary problems (10, 11, 12, 13, 14). The mutations occur in the DNA-binding domain (11, 12), the N-terminal domain (10, 11) or intron 2 (13, 14). In all cases, the DNA-binding domain is altered or missing with parallel loss of functional activity, producing a reduction by half in the TTF1 protein levels, a phenomenon known as haploinsufficiency (45).

In the patients reported here, haploinsufficiency may also be the mechanism involved because, although the mutation is not in the DNA binding domain, it does generate a protein that has lost its transactivation capacity. Furthermore, the transfection data point to a tissue-specific dominant-negative effect. In fact, it has been suggested that if a deletion changes only the carboxy terminus of a transcription factor, the mut may function as a poison protein, interfering with the activity of the wt protein (45).

To our knowledge, only one C-terminal domain mutation has been described in TTF1 (12), which is a frameshift mutation that results in an aberrant protein starting at codon 273 [named 303 by Breedveld et al. (12)], rather than 275; this protein also lacked 22 amino acids. The patient had BHC, although the status of respiratory and thyroid function was not reported, and no functional assays were carried out.

We predicted the secondary structure of the nonsense region generated in the mutated protein and compared it with the wt protein. The SOPMA program (36) predicts a secondary structure with a greatly altered C-terminal domain in the mutated protein (Fig. 8Go). The mut protein has mainly a random coil structure, interrupted by a single helix in a position that differs from that of wt TTF1. These data were confirmed by several different secondary structure prediction methods (46, 47). The properties of this structure could affect DNA binding, transactivation, or protein-protein interactions. We performed experiments to elucidate which of the above properties is affected. Our results show that the DNA binding capability of the mutated protein is reduced and that it does not interfere with the DNA binding of the wt protein. We show, however, that the mutated protein has a strong dominant-negative effect on transcription from the Tg and TPO promoters but not from the SP-B promoter. The differential effect may indicate that the mutated protein impairs the interaction of wt TTF1 with different activators or coactivators in thyroid but not in lung. Actually, TTF1 interacts with various factors in a tissue-specific manner, such as PAX8 and DREAM in thyroid (37, 48) and AP-1 family members, BR22, RARs, CBP/p300, SRC-1, GATA-6, STAT-3, NF-1, and TAZ in lung (49, 50, 51, 52, 53, 54, 55, 56). We present preliminary evidence, using both the Tg and artificial Gal4-binding-site promoters, that mutated TTF1 interferes with the cooperative interaction between wt TTF1 and PAX8.


Figure 8
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  		FIG. 8. Analysis of the secondary structure of the C-terminal region of TTF1. Prediction was done from the mutated codon (275) to the end for both functional and nonsense proteins, using the SOPMA method (36 ). Note that the sequences generate two completely different structures. c, Random coil; h, {alpha}-helix; e, extended strand; t, ß-turn; aa, amino acids.

 
An intriguing question is why we observed the dominant-negative effect on the Tg and TPO promoters in HeLa cells that do not express PAX8. This observation may indicate that other proteins with a similar activity replace PAX8 function in HeLa cells. Although additional experiments regarding this issue remain to be performed, we believe that this piece of information is of interest to understand the phenotype of the two sisters studied in this work. Another possible explanation for the differential role of the mutated TTF1 protein in thyroid and lung may be related to the different architecture of the Tg, TPO (18, 57), or SP-B (29) promoters with which TTF1 interacts. Because promoter sequences provide a platform for a large number of direct or indirect proteins interactions, the consequences of reduced binding of even one transcription factor could be considerable. Besides, the loss of one transactivation domain, two serines, necessary for TTF1 phosphorylation (39), and one cysteine, involved in TTF1 dimerization (40), may affect specific, complex mechanisms such as cell type-specific selection of target promoters.

The neurological mechanisms responsible for BHC are still poorly understood. This disease is caused by haploinsufficiency of the TTF1 protein. In mouse brain, TTF1 is expressed in the median ganglionic eminence, which gives rise to the pallidal component of the basal ganglia. Consistent with this expression pattern, TITF1/NKX2.1 null mice do not form the pallidal structures due to ventral-to-dorsal transformation of the pallidal primordium into a striatum-like structure (22, 58). These mice show an imbalance in the number of striatal and pallidal neurons, which could be responsible for hyperkinesia (10) and choreoathetotic movements (11). In the sisters studied here, the choreoathetotic phenotype can be explained by the mutation reported; we speculate that an increased number of striatal neurons could be the cause.

In conclusion, our functional data explain that the phenotype observed in two patients with hypothyroidism but no pulmonary distress is due to a deletion of cytosine 825 in exon 3 of the TITF1/NKX2.1 gene. Experiments are nonetheless required to understand the role of the TITF1/NKX2.1 gene at the neurological level and clarify the mechanisms involved in the choreoathetotic phenotype.


   Acknowledgments
 
We thank all members of the affected families for their collaborative participation in this study. We are grateful to Dr. Samuel. Refetoff (University of Chicago, Chicago, IL) for human pcDNA3TTF1, pTGenh/prom-Luc, and hTPOprom-Luc; Dr. J. Jeffrey A. Witsett (Children’s Hospital Medical Center, Cincinnati, OH) for hSP-Bprom-Luc; Dr. Gilbert Vassart (Université Libre de Bruxelles, Brussels, Belgium) for human Pax8 expression vector; and Drs. Roberto Di Lauro and Mario De Felice (Stazione Zoologica Anthor Dohrn, Naples, Italy) for pG5 vector. We also thank members of Dr. Santisteban’s laboratory for advice and comments, and G. Riesco-Eizaguirre, M.D. (Hospital La Paz and Instituto de Investigaciones Biomédicas, Madrid, Spain) for critical reading of the manuscript. We are indebted to Dr. Rodrigo Jacamo (David Geffen School of Medicine, Department of Medicine, Division of Digestive Disease, University of California, Los Angeles, Los Angeles, CA) for scientific and technical advice on the site-directed mutagenesis strategy. We thank Dr. Ronald Hartong for his criticism and his linguistic assistance.


   Footnotes
 
This work was supported by grants from the Dirección General de Investigación BFU2004-03169 (Ministerio Educación y Ciencia), Fondo Investigaciones Sanitarias (FIS) of the Instituto de Salud Carlos III Red Grupos Diabetes Mellitus (C03/212), Red Centro Metabolismo Nutrición (C03/08), and PI041216, Comunidad Autónoma Madrid GR/SAL/0773/2004 and Pfizer (Spain). C.M.M is a recipient of a postdoctoral fellowship from the Oncology Program of the Fundación Carolina (Spain). G.P.d.N. and J.R.B. are FIS Research Scientists supported by the Spanish Ministry of Health (Fellowships CP03/0064 and 99/3076, respectively).

We have nothing to declare.

First Published Online February 28, 2006

1 C.M.M. and G.P.d.N. contributed equally to this work and both should be considered first authors. Back

Abbreviations: BHC, Benign hereditary chorea; CH, congenital hypothyroidism; LUC, luciferase; mut, mutant; PAX8, paired box 8; Sp1, specificity protein-1; SP-B, surfactant protein B; Tg, thyroglobulin; TPO, thyroperoxidase; TTF1, thyroid transcription factor 1; TITF1/NKX2.1, thyroid transcription factor 1 gene; wt, wild-type.

Received July 6, 2005.

Accepted February 16, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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