AUTHOR=Delestrain Céline , Aissat Abdel , Nattes Elodie , Gibertini Isabelle , Lacroze Valérie , Simon Stéphanie , Decrouy Xavier , de Becdelièvre Alix , Fanen Pascale , Epaud Ralph TITLE=Deciphering an isolated lung phenotype of NKX2-1 frameshift pathogenic variant JOURNAL=Frontiers in Pediatrics VOLUME=10 YEAR=2023 URL=https://www.frontiersin.org/journals/pediatrics/articles/10.3389/fped.2022.978598 DOI=10.3389/fped.2022.978598 ISSN=2296-2360 ABSTRACT=Background

to perform a functional analysis of a new NK2 homeobox 1 (NKX2-1) variant (c.85_86del denominated NKX2-1DEL) identified in a family presenting with isolated respiratory disease, in comparison to another frameshift variant (c.254dup denominated NKX2-1DUP) identified in a subject with classical brain-lung-thyroid syndrome.

Methods

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 SFTPA, SFTPB, SFTPC, and ABCA3 promoters was assessed in A549 cells. Thyroglobulin promoter activity was measured with the paired box gene 8 (PAX8) cofactor in Nthy cells.

Results

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 SFTPA, SFTPB, SFTPC, ABCA3 and thyroglobulin promoters was significantly decreased with both variants. However, with NKX2-1DEL, thyroglobulin transcriptional activity was maintained with the addition of PAX8.

Conclusion

These results provide novel insights into understanding the molecular mechanism of phenotypes associated with NKX2-1 pathogenic variants.