A Novel Missense Variant in the NKX2-1 Homeodomain Prevents Transcriptional Rescue by TAZ.

Background: Brain-lung-thyroid syndrome (BLTS) is caused by NKX2-1 haploinsufficiency, resulting in chorea/choreoathetosis, respiratory problems, and hypothyroidism. Genes interacting with NKX2-1 mutants influence its phenotypic variability. We report a novel NKX2-1 missense variant and the modifier function of TAZ/WWTR1 in BLTS. Methods: A child with BLTS underwent next-generation sequencing panel testing for thyroid disorders. His family was genotyped for NKX2-1 variants and screened for germline mosaicism. Mutant NKX2-1 was generated, and transactivation assays were performed on three NKX2-1 target gene promoters. DNA binding capacity and protein-protein interaction were analyzed. Results: The patient had severe BLTS and carried a novel missense variant c.632A>G (p.N211S) in NKX2-1, which failed to bind to specific DNA promoters, reducing their transactivation. TAZ cotransfection did not significantly increase transcription of these genes, although the variant retained its ability to bind to TAZ. Conclusions: We identify a novel pathogenic NKX2-1 variant that causes severe BLTS and is inherited through germline mosaicism. The mutant lacks DNA-binding capacity, impairing transactivation and suggesting that NKX2-1 binding to DNA is essential for TAZ-mediated transcriptional rescue.

TAZ/WWTR1 Mediates the Pulmonary Effects of NKX2-1 Mutations in Brain-Lung-Thyroid Syndrome.

Context: Identification of a frameshift heterozygous mutation in the transcription factor NKX2-1 in a patient with brain-lung-thyroid syndrome (BLTS) and life-threatening lung emphysema. Objective: To study the genetic defect that causes this complex phenotype and dissect the molecular mechanism underlying this syndrome through functional analysis. Methods: Mutational study by DNA sequencing, generation of expression vectors, site-directed mutagenesis, protein-DNA-binding assays, luciferase reporter gene assays, confocal microscopy, coimmunoprecipitation, and bioinformatics analysis. Results: We identified a mutation [p.(Val75Glyfs*334)] in the amino-terminal domain of the NKX2-1 gene, which was functionally compared with a previously identified mutation [p.(Ala276Argfs*75)] in the carboxy-terminal domain in other patients with BLTS but without signs of respiratory distress. Both mutations showed similar protein expression profiles, subcellular localization, and deleterious effects on thyroid-, brain-, and lung-specific promoter activity. Coexpression of the coactivator TAZ/WWTR1 (transcriptional coactivator with PDZ-binding motif/WW domain-containing transcription regulator protein 1) restored the transactivation properties of p.(Ala276Argfs*75) but not p.(Val75Glyfs*334) NKX2-1 on a lung-specific promoter, although both NKX2-1 mutants could interact equally with TAZ/WWTR1. The retention of residual transcriptional activity in the carboxy-terminal mutant, which was absent in the amino-terminal mutant, allowed the functional rescue by TAZ/WWTR1. Conclusions: Our results support a mechanistic model involving TAZ/WWTR1 in the development of human congenital emphysema, suggesting that this protein could be a transcriptional modifier of the lung phenotype in BLTS.