Thyroid Molecular Laboratory, Institute for Medical and Molecular Genetics, La Paz University Hospital, Madrid, Spain.
Biomedical Research Institute "Alberto Sols," Spanish National Council for Scientific Research-Autonomous University of Madrid (CSIC-UAM), Madrid, Spain.
J Clin Endocrinol Metab. 2018 Mar 1;103(3):839-852. doi: 10.1210/jc.2017-01241.
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.
To study the genetic defect that causes this complex phenotype and dissect the molecular mechanism underlying this syndrome through functional analysis.
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.
We identified a mutation [p.(Val75Glyfs334)] in the amino-terminal domain of the NKX2-1 gene, which was functionally compared with a previously identified mutation [p.(Ala276Argfs75)] 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.(Ala276Argfs75) but not p.(Val75Glyfs334) 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.
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.
在患有脑肺甲状腺综合征（BLTS）和危及生命的肺气肿的患者中，鉴定出转录因子 NKX2-1 的杂合移码突变。
研究导致这种复杂表型的遗传缺陷，并通过功能分析剖析该综合征的分子机制。
通过 DNA 测序进行突变研究、生成表达载体、定点诱变、蛋白-DNA 结合测定、荧光素酶报告基因测定、共聚焦显微镜、共免疫沉淀和生物信息学分析。
我们在 NKX2-1 基因的氨基末端结构域中发现了一个突变[p.(Val75Glyfs334)]，与其他患有 BLTS 但没有呼吸窘迫迹象的患者中羧基末端结构域的先前鉴定的突变[p.(Ala276Argfs75)]进行了功能比较。这两种突变均显示出相似的蛋白表达谱、亚细胞定位，以及对甲状腺、脑和肺特异性启动子活性的有害影响。共表达共激活因子 TAZ/WWTR1（具有 PDZ 结合基序/WW 结构域包含转录调节剂蛋白 1 的转录共激活因子）恢复了 p.(Ala276Argfs75)的转录激活特性，但 p.(Val75Glyfs334) NKX2-1 则没有，尽管两种 NKX2-1 突变体都可以与 TAZ/WWTR1 同等相互作用。在羧基末端突变体中保留的残留转录活性在氨基末端突变体中不存在，这允许 TAZ/WWTR1 进行功能挽救。
我们的结果支持涉及 TAZ/WWTR1 的机制模型在人类先天性肺气肿的发生发展中，表明该蛋白可能是 BLTS 中肺表型的转录调节剂。
