Delestrain Céline, Aissat Abdel, Nattes Elodie, Gibertini Isabelle, Lacroze Valérie, Simon Stéphanie, Decrouy Xavier, de Becdelièvre Alix, Fanen Pascale, Epaud Ralph
Univ Paris Est Creteil, INSERM, IMRB, Creteil, France.
Centre Hospitalier Intercommunal de Créteil, Service de Pédiatrie Générale, Créteil, France.
Front Pediatr. 2023 Jan 17;10:978598. doi: 10.3389/fped.2022.978598. eCollection 2022.
to perform a functional analysis of a new NK2 homeobox 1 (NKX2-1) variant (c.85_86del denominated NKX2-1) identified in a family presenting with isolated respiratory disease, in comparison to another frameshift variant (c.254dup denominated NKX2-1) identified in a subject with classical brain-lung-thyroid syndrome.
pathogenic variants were introduced into the pcDNA3-1(+)-wt-TTF1 plasmid. The proteins obtained were analyzed by western blot assay. Subcellular localization was assessed by confocal microscopy in A549 and Nthy cells. Transactivation of , , , and promoters was assessed in A549 cells. promoter activity was measured with the paired box gene 8 (PAX8) cofactor in Nthy cells.
The two sequence variants were predicted to produce aberrant proteins identical from the 86th amino acid, with deletion of their functional homeodomain, including the nuclear localization signal. However, 3D conformation prediction of the conformation prediction of the mutant protein assumed the presence of a nuclear localization signal, a bipartite sequence, confirmed by confocal microscopy showing both mutant proteins localized in the nucleus and cytoplasm. Transcriptional activity with and promoters was significantly decreased with both variants. However, with NKX2-1, transcriptional activity was maintained with the addition of PAX8.
These results provide novel insights into understanding the molecular mechanism of phenotypes associated with pathogenic variants.
对在一个患有孤立性呼吸系统疾病的家族中鉴定出的一种新的NK2同源盒1（NKX2-1）变体（命名为NKX2-1的c.85_86del）进行功能分析，并与在一名患有经典脑-肺-甲状腺综合征的受试者中鉴定出的另一种移码变体（命名为NKX2-1的c.254dup）进行比较。
将致病变体引入pcDNA3-1(+)-wt-TTF1质粒。通过蛋白质免疫印迹分析对获得的蛋白质进行分析。通过共聚焦显微镜在A549和Nthy细胞中评估亚细胞定位。在A549细胞中评估、、和启动子的反式激活。在Nthy细胞中用配对盒基因8（PAX8）辅因子测量启动子活性。
预测这两个序列变体从第86个氨基酸开始产生相同的异常蛋白质，其功能性同源结构域缺失，包括核定位信号。然而，突变蛋白的三维构象预测假定存在一个核定位信号，这是一个二分序列，共聚焦显微镜证实这两种突变蛋白都定位于细胞核和细胞质中。两种变体与和启动子的转录活性均显著降低。然而，对于NKX2-1，添加PAX8后转录活性得以维持。
这些结果为理解与致病变体相关的表型的分子机制提供了新的见解。
