Background: NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS). Methods: The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. Results: The mutation is a deletion of a cytosine at position 834 (ref. sequence NM-003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. Conclusions: We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype-phenotype correlations in the NKX2-1 deficiency syndrome. © Copyright 2013, Mary Ann Liebert, Inc. 2013.

Identification and functional characterization of a novel mutation in the NKX2-1 gene: Comparison with the data in the literature / Nettore, I. C.; Mirra, P.; Ferrara, A. M.; Sibilio, A.; Pagliara, V.; Kay, C. S. K.; Lorenzoni, P. J.; Werneck, L. C.; Bruck, I.; Dos Santos, L. H. C.; Beguinot, F.; Salvatore, D.; Ungaro, P.; Fenzi, G.; Scola, R. H.; Macchia, P. E.. - In: THYROID. - ISSN 1050-7256. - 23:6(2013), pp. 675-682. [10.1089/thy.2012.0267]

Identification and functional characterization of a novel mutation in the NKX2-1 gene: Comparison with the data in the literature

Nettore I. C.;Mirra P.;Ferrara A. M.;Pagliara V.;Beguinot F.;Salvatore D.;Ungaro P.;Fenzi G.;Macchia P. E.
2013

Abstract

Background: NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS). Methods: The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. Results: The mutation is a deletion of a cytosine at position 834 (ref. sequence NM-003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. Conclusions: We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype-phenotype correlations in the NKX2-1 deficiency syndrome. © Copyright 2013, Mary Ann Liebert, Inc. 2013.
2013
Identification and functional characterization of a novel mutation in the NKX2-1 gene: Comparison with the data in the literature / Nettore, I. C.; Mirra, P.; Ferrara, A. M.; Sibilio, A.; Pagliara, V.; Kay, C. S. K.; Lorenzoni, P. J.; Werneck, L. C.; Bruck, I.; Dos Santos, L. H. C.; Beguinot, F.; Salvatore, D.; Ungaro, P.; Fenzi, G.; Scola, R. H.; Macchia, P. E.. - In: THYROID. - ISSN 1050-7256. - 23:6(2013), pp. 675-682. [10.1089/thy.2012.0267]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/838282
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