A Novel Mutation in NKX2-1 Shows Dominant-Negative Effects Only in the Presence of PAX8

Abstract

To date, >100 mutations in NKX2-1 have been described. Most NKX2-1 mutations are assumed to result in brain-lung-thyroid syndrome through haploinsufficiency, and only five NKX2-1 mutations with dominant-negative effects have been reported so far. In this case report, an additional patient with brain-lung-thyroid syndrome is reported, carrying a novel heterozygous mutation, c.533G>C (p.R178P), in the homeobox of NKX2-1. This mutation has been proven to be a dominant-negative mutation by an in vitro functional assay. Of note, the dominant-negative effect of R178P-NKX2-1 was shown only in the presence of PAX8.

Introduction

Brain-lung-thyroid syndrome (BLTS; OMIM #610978) is an autosomal dominant disorder characterized by choreoathetosis, congenital hypothyroidism (CH), and pulmonary dysfunction in infancy. This syndrome results from heterozygous mutations in NKX2-1 (OMIM #600635), which encodes a transcription factor with a significant impact on the development of the brain, lung, and thyroid. Here, a familial case of BLTS is reported where a novel heterozygous missense mutation, c.533G>C (p.R178P), was identified in the homeobox of NKX2-1, which resulted in a dominant-negative effect. Of note, the dominant-negative effect of R178P-NKX2-1 was shown only in the presence of PAX8.

Patients

The proband was a seven-year-old girl born at 40 weeks of gestation after an uncomplicated pregnancy and delivery. Her birth weight was 2750 g (−0.6 SD). She was diagnosed as having subclinical hypothyroidism (thyrotropin [TSH] level >9 μIU/mL) by neonatal screening. Her serum TSH level decreased to 6.3 μIU/mL at the age of 15 days, and ultrasonography showed a normal-sized thyroid gland. Therefore, replacement therapy was not started. Although she then was biochemically euthyroid, she showed slightly delayed psychomotor development. At the age of two years and four months, her thyroid function was reevaluated. She had a slightly elevated serum TSH level (19.0 mIU/L) with normal thyroid hormone levels (free thyroxine [fT4] = 0.96 ng/dL). Levothyroxine supplementation was started for compensated hypothyroidism. At the age of three years, she developed choreoathetosis. She required repeated hospitalizations due to recurrent lower respiratory infections and asthma attacks during infancy.

The patient's father is currently 28 years old and had motor development delays in infancy, as well as gait abnormalities in childhood, which improved by the age of six years. He also suffered from recurrent respiratory infections in infancy. At the age of 28 years, he was diagnosed for the first time with subclinical hypothyroidism (TSH = 8.13 = μIU/mL; fT4 = 0.74 ng/dL; free triiodothyronine = 3.70 pg/mL).

Results

The study was approved by the Institutional Review Board of the Tokyo Metropolitan Children's Medical Center (H24-68), and informed consent was obtained from the parents. The two coding exons and flanking introns of NKX2-1 (NM_003317) were analyzed, and a novel heterozygous c.533G>C (p.R178P) mutation was found in the proband and her affected father. The mutation is located in the homeobox of NKX2-1 (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/thy). Arginine 178 is highly evolutionarily conserved in all vertebrate NKX2s (Supplementary Fig. S1B). This mutation was not detected in any of the 150 healthy controls, and was absent from databases such as dbSNP, the 1000 Genomes Project, Exome Variant Server, NHLBI Exome Sequencing Project, and the Human Genetic Variation Database (HGVD) in Japan. The in silico mutation analysis tools SIFT and PolyPhen-2 generated scores of 0.00 (deleterious) and 0.999 (probably damaging).

The details of the experimental procedures of functional studies are described in the Supplementary Methods. Western blotting and subcellular localization studies showed that there were no significant differences between the protein expression levels in wild-type (WT) and mutant NKX2-1 (Fig. 1A and B). WT-NKX2-1 stimulated the transcription of a TG-luc reporter in a dose-dependent manner. PAX8 had synergistic transactivation properties on the TG promoter with WT-NKX2-1. Stimulation with the R178P-NKX2-1 showed markedly reduced transactivation in the presence or absence of PAX8 or P300 (Fig. 1C). The dominant-negative effect of R178P-NKX2-1 occurred only in the presence of PAX8. The electrophoretic mobility shift assays experiment showed that WT-NKX2-1 specifically bound to DNA, and this binding was completed by 200-fold excess cold competitor. In contrast, R178P-NKX2-1 had abrogated DNA-binding ability (Fig. 1D). The pull-down assay revealed that the binding efficiency between R178P-NKX2-1 and PAX8 was comparable to that between WT-NKX2-1 and PAX8 (Fig. 1E). A transactivation assay was also performed using the lung surfactant protein promoter (SFTPB). WT-NKX2-1 stimulated the transcription of a SFTPB-luc reporter in a dose-dependent manner. The dominant-negative effect of R178P-NKX2-1 was not shown on the SFTPB promoter (Supplementary Fig. S2).

FIG. 1.

Functional characterization of R178P-NKX2-1. (A) Western blot analysis. There was no significant difference between the protein expression levels in wild-type (WT)-NKX2-1 and R178P-NKX2-1. (B) Subcellular localization analysis. Both the WT-NKX2-1 and R178P-NKX2-1 were localized in the nucleus. (C) Transactivation assays. HEK293 cells were co-transfected with the pRL-CMV internal control vector, the indicated amount (in nanograms) of the effector plasmids (WT-NKX2-1, R178P-NKX2-1, PAX8, and P300), and the TG-Luc reporter. Transactivation by WT-NKX2-1 (black bars) and R178P-NKX2-1 (striped bars) co-transfected with WT and R178P-NKX2-1 (gray bars) on the TG-luc reporter plasmid via interaction with or without PAX8 or P300. WT-NKX2-1 stimulated the transcription of TG-luc reporter in a dose -dependent manner. PAX8 synergized with WT-NKX2-1, but did not rescue the transactivation of R178P-NKX2-1. R178P-NKX2-1 showed markedly reduced transactivation on the TG-luc reporter. R178P-NKX2-1 interfered with the transcriptional activity of WT-NKX2-1, indicating a dominant-negative effect, in the presence of PAX8, but this did not occur in the absence of PAX8, even with increased amounts of mutated protein. The data shown represent the mean ± standard error of the mean of at least three independent experiments (each performed in triplicate). *p < 0.05; **p < 0.01. (D) Electrophoretic mobility shift assay experiment. WT-NKX2-1 specifically bound to DNA and this binding was completed by 200-fold excess cold competitor. In contrast, R178P-NKX2-1 had abrogated DNA-binding ability. (E) Pull-down assay. The binding efficiency between R178P-NKX2-1 and PAX8 was comparable to that between WT- NKX2-1 and PAX8. Color images available online at www.liebertpub.com/thy

Discussion

To date, >100 mutations of NKX2-1 have been described, and most NKX2-1 mutations are assumed to cause BLTS through haploinsufficiency. A nonsense mutation affecting R178 (p.R178*) has already been reported by Provenzano et al. (1). The authors showed that R178*-NKX2-1 was not efficiently translocated into the nucleus. Consequently, it could not interfere with the transcriptional activity of the WT protein, and therefore it had no dominant-negative effect. To date, only five NKX2-1 mutations with dominant-negative effects (R165fs, L176V, I207F, L263fs, and P275fs) have been reported (2 –6). Among them, R165fs and P275fs have shown to result in dominant-negative effects on the TG promoter but not the lung surfactant protein promoters (2,6). On the other hand, L263fs had a dominant effect on the lung surfactant protein promoters (SFTPB and SFTPC) but not on the TG promoter (5). These results demonstrating promoter or tissue-specific dominant-negative effects strongly support the significance of the cofactors, such as PAX8, TAZ, and P300, in the mechanism of dominant-negative effects. All four mutant NKX2-1 (R165fs, L176V, I207F, and P275fs) showed dominant-negative effects without PAX8 co-transfection. Therefore, R178P-NKX2-1, which showed dominant-negative effects only in the presence of PAX8, is the first example with clear mechanistic involvement of PAX8 in the dominant-negative effects on the TG promoter. A pull-down assay revealed that the binding efficiency between R178P-NKX2-1 and PAX8 was comparable to that between WT and PAX8. Therefore, R178P-NKX2-1 causes a dominant-negative effect, possibly due to competition with PAX8. This finding is novel and extends our understanding of the complex molecular and pathogenic mechanisms associated with the mutations in NKX2-1 and PAX8.

Footnotes

Acknowledgments

The authors thank Dr. S. Narumi (Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan) for the TG-Luc reporter construct. This work was supported by a Grant from the Japan Society for the Promotion of Science (16K10007), Takeda Science Foundation, and the Foundation for Growth Science, Japan.

Author Disclosure Statement

The authors have nothing to disclose.

References

Provenzano

, Veneziano

, Appleton

, Frontali

, Civitareale

. 2008. Functional characterization of a novel mutation in TITF-1 in a patient with benign hereditary chorea. J Neurol Sci, 264:56–62.

Nettore

, Mirra

, Ferrara

, Sibilio

, Pagliara

, Kay

, Lorenzoni

, Werneck

, Bruck

, Dos Santos

, Beguinot

, Salvatore

, Ungaro

, Fenzi

, Scola

, Macchia

. 2013. Identification and functional characterization of a novel mutation in the NKX2-1 gene: comparison with the data in the literature. Thyroid, 23:675–682.

Carré

, Szinnai

, Castanet

, Sura-Trueba

, Tron

, Broutin-L'Hermite

, Barat

, Goizet

, Lacombe

, Moutard

, Raybaud

, Raynaud-Ravni

, Romana

, Ythier

, Leger

, Polak

. 2009. Five new TTF1/NKX2.1 mutations in brain-lung-thyroid syndrome: rescue by PAX8 synergism in one case. Hum Mol Genet, 18:2266–2276.

Maquet

, Costagliola

, Parma

, Christophe-Hobertus

, Oligny

, Fournet

, Robitaille

, Vuissoz

, Payot

, Laberge

, Vassart

, Van Vliet

, Deladoey

. 2009. Lethal respiratory failure and mild primary hypothyroidism in a term girl with a de novo heterozygous mutation in the NKX2-1/NKX2.1 gene. J Clin Endocrinol Metab, 94:197–203.

Guillot

, Carré

, Szinnai

, Castanet

, Tron

, Jaubert

, Broutin

, Counil

, Feldmann

, Clement

, Polak

, Epaud

. 2010. NKX2-1 mutations leading to surfactant protein promoter dysregulation cause interstitial lung disease in “Brain-Lung-Thyroid Syndrome.”. Hum Mutat, 31: 1146–1162.

Moya

, Perez de Nanclares

, Castano

, Potau

, Bilbao

, Carrascosa

, Bargada

, Coya

, Martul

, Vicens-Calvet

, Santisteban

. 2006. Functional study of a novel single deletion in the NKX2-1/NKX2.1 homeobox gene that produces congenital hypothyroidism and benign chorea but not pulmonary distress. J Clin Endocrinol Metab, 91:1832–1841.