Deciphering an isolated lung phenotype of NKX2-1 frameshift pathogenic variant

Abstract

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

Keywords: NKX2-1; child; lung; surfactant protein; thyroid.

Figure 1

(A) Identification of two NKX2-1 pathogenic variant by direct sequencing and expression of the wild-type and mutated NKX2-1 protein. The diagram of the NKX2-1 transcript variant 2 (NM_003317.3) shows the position of the homeodomain and variants. (B) Deduced amino acid sequences of NKX2-1^dup and NKX2-1^del as compared with NKX2-1^WT. The nuclear localization signal (NLS) is underlined in blue for the NKX2-1^WT and the putative NLSs, according to the predictions, are underlined in red for the two mutants. The homeobox domain is framed in gray (position 160 to 220) for the NKX2-1^WT and is absent for both mutants. pSP motifs have been highlighted in light blue. The NKX2-1^WT harbors five motifs while NKX2-1^del and NKX2-1^dup have three motifs. LPPY motif, a putative TAZ protein interaction motif, has been highlighted in orange for the NKX2-1-^WT. This motif is absent for both mutants. Serines involved in phosphorylation in the wild-type sequence but affected by the mutations are marked by an asterisk. Cysteines involved in the dimerization of the amino-acid chains in the wild-type but affected by the mutations are marked by a plus sign (adapted from Moya et al., 2018). (C) Representative western blot analysis in pcDNA3-1(+)-TTF1 plasmid of translated NKX2-1 wild type and mutant (DEL and DUP). (D) Representative immunoblotting of NKX2-1 in whole cell lysates of A549 cells not transfected (Control) or transiently transfected with NKX2-1^WT, NKX2-1^DUP and NKX2-1^DEL. (E) Representative immunoblotting of NKX2-1 in whole cell lysates of Nthy cells non-transfected (Control) or transiently transfected with NKX2-1^WT, NKX2-1^DUP and NKX2-1^DEL.

Figure 2

Representative confocal images of A549 cells (A) and nthy cells (B) after 24-h expression of NKX2-1^WT, NKX2-1^DEL and NKX2-1^DUP. Immunostaining involved incubation with rabbit polyclonal anti-NKX2-1 antibody (green). Nuclei were detected by ToPro-3 iodide (blue). Scatter graphs show the nucleocytoplasmic ratio of each protein (≥10 cells per experiment, n = 3 or 4).

Figure 3

Functional activity of wild-type vs. mutant NKX2-1 on promoters of lung-specific genes (SFTPC, SFTPB, ABCA3 and SFTPA). Transactivation of SFTPA (A), SFTPB (B), SFTPC (C), and ABCA3 (D) by wild-type NKX2-1, NKX2-1^DUP or NKX2-1^DEL. Promoter constructs were co-transfected in A549 cells (A–D) with the empty pcDNA3.1 vector or expression vector for the wild-type or mutated NKX2-1 cDNAs. Data are mean ± SEM of five independent experiments performed in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Functional activity of wild-type vs. mutants NKX2-1 on thyroid-specific (thyroglobulin) gene. Transactivation of the thyroglobulin promoter by NKX2-1^WT, NKX2-1^DUP or NKX2-1^DEL. Promoter constructs were co-transfected without (A) or with (B) PAX8 in Nthy cells with the empty pcDNA3.1 vector or the expression vector for wild-type or mutated NKX2-1 cDNAs. Data are mean ± SEM of five independent experiments performed in duplicate. ***p < 0.001.