Moya Christian M, Perez de Nanclares Guiomar, Castaño Luis, Potau Neus, Bilbao J Ramón, Carrascosa Antonio, Bargadá María, Coya Raquel, Martul Pedro, Vicens-Calvet Enric, Santisteban Pilar
Biomedical Research Institute Alberto Sols, Spanish National Research Council-Autonomous University of Madrid, Madrid, Spain.
J Clin Endocrinol Metab. 2006 May;91(5):1832-41. doi: 10.1210/jc.2005-1497. Epub 2006 Feb 28.
We studied two sisters with congenital hypothyroidism and choreoathetosis but not respiratory distress.
The aim of this study was to establish the genetic defect that causes this phenotype and study the molecular mechanisms of the pathology by means of functional analysis.
Sequencing of DNA, expression vectors generation, EMSAs, transfections experiments as well as bioinformatics analysis were performed.
We found a new single deletion (825delC) in one allele of the TITF1/NKX2.1 gene. The mutation located in the C-terminal domain generates a nonsense thyroid transcription factor 1 (TTF1) protein, with 22 amino less and rich in positive charges. This protein shows diminished binding to DNA, does not interfere with wild-type (wt) TTF1 binding, and fails to activate reporter genes harboring the thyroglobulin (Tg), thyroperoxidase (TPO), or surfactant protein B (SP-B) promoters. In addition, the mutant (mut) protein has a dominant-negative effect on the transcriptional activity of wt TTF1 in a promoter-specific manner, inhibiting the transcription of Tg and TPO but not of SP-B. Using a Gal4 reporter system, we demonstrate that the mut protein is not transcriptionally active and does not likely compete with the wild type for coactivators. Interestingly, the mut protein impairs the wt capacity to synergize with paired box 8 (PAX8). This cooperation is necessary for Tg and TPO transcription but dispensable for SP-B expression.
These results are concordant with the phenotype of the two sisters studied and demonstrate a differential role for TTF1 in the different tissues in which it is expressed.
我们研究了两名患有先天性甲状腺功能减退症和舞蹈手足徐动症但无呼吸窘迫症状的姐妹。
本研究旨在确定导致这种表型的基因缺陷，并通过功能分析研究病理的分子机制。
进行了DNA测序、表达载体构建、电泳迁移率变动分析（EMSA）、转染实验以及生物信息学分析。
我们在TITF1/NKX2.1基因的一个等位基因中发现了一个新的单碱基缺失（825delC）。该突变位于C末端结构域，产生一种无义甲状腺转录因子1（TTF1）蛋白，其氨基酸减少了22个且富含正电荷。这种蛋白与DNA的结合能力减弱，不干扰野生型（wt）TTF1的结合，并且无法激活含有甲状腺球蛋白（Tg）、甲状腺过氧化物酶（TPO）或表面活性蛋白B（SP-B）启动子的报告基因。此外，突变（mut）蛋白对wt TTF1的转录活性具有启动子特异性的显性负效应，抑制Tg和TPO的转录，但不抑制SP-B的转录。使用Gal4报告系统，我们证明mut蛋白没有转录活性，并且不太可能与野生型竞争共激活因子。有趣的是，mut蛋白损害了wt与配对盒8（PAX8）协同作用的能力。这种协同作用对于Tg和TPO的转录是必需的，但对于SP-B的表达是可有可无的。
这些结果与所研究的两姐妹的表型一致，并证明了TTF1在其表达的不同组织中的不同作用。
