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

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 in a Brazilian family with clinical features of BTLS was sequenced 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 (NLS), the whole homeodomain and the carboxy-terminus of NKX2-1 are all missing in the mutant protein that 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 and the Surfactant Protein-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 is also noticed when the mutation is tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared to 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.