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  • Clinical Research Article
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Functional characterization of two novel NKX2-1 frameshift variants that cause pulmonary surfactant dysfunction

Pediatric Research volume 95, pages 744–751 (2024)Cite this article

Abstract

Background

We aim to report two unrelated patients with pulmonary surfactant dysfunction (PSD) that carried two novel NKX2-1 frameshift variants, and evaluated the impact of these variants in vitro.

Methods

We enrolled children with PSD and NKX2-1 variants, and collected their clinical information and follow-up data. We constructed wild-type (WT) and variant NKX2-1 plasmids and transfected them into A549 and HEK293T cells. The functional characterization of variants was then evaluated by qRT-PCR, western blot, immunofluorescence, electrophoretic mobility shift assay, and dual-luciferase reporter assay.

Results

Two novel heterozygous frameshift variants of NKX2-1, i.e., c.705delC (Gly236Alafs*29) and c.313_316 dup (Asn106Lysfs*304), were identified in children from two unrelated families. We discerned attenuated mRNA and protein expression in the Asn106Lysfs*304 variant, and reduced DNA -binding as well as transcriptional activation capabilities in both variants. While the Asn106Lysfs*304 variant lost its synergistic interactions with PAX8 and TAZ, the Gly236Alafs*29 variant partially retained its residual transcriptional activation capabilities and synergistic interactions with PAX8 and TAZ.

Conclusions

We reported on two children with two novel NKX2-1 frameshift variants. In vitro experiments revealed that the two frameshift variants have common and different mechanisms based on the loss or conservation of domains, which partially explained the phenotypical heterogeneity.

Impact

  • Pulmonary surfactant dysfunction is a rare heterogeneous disease that exhibits a great burden on children’s quality of life.

  • We reported two novel NKX2-1 frameshift variants carried by two children with different clinical phenotypes, thus broadening our knowledge base of gene variations and phenotypes in NKX2-1.

  • We performed an in vitro study and uncovered different pathogenic mechanisms underlying the actions of two novel variants, and thereby partially explained the mechanisms of phenotypical heterogeneity caused by NKX2-1 variants.

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Fig. 1: Clinical information of patients and NKX2-1 variants genetic analysis.
Fig. 2: mRNA and protein expression levels, nuclear translocation, and DNA-binding abilities of NKX2-1 variants.
Fig. 3: The transcriptional transactivation and synergistic interaction between the variants and TAZ or PAX8.

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Acknowledgements

The authors would like to thank all the patients and their families involved in this study.

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 82171707) and the Key Projects of the Natural Science Foundation from Fujian Provincial Department of Science and Technology (No. 2022D012).

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Author notes

  1. These authors contributed equally: Huixian Wang, Gaoli Jiang.

Authors and Affiliations

  1. Division of Pulmonary Medicine, Children’s Hospital of Fudan University, Shanghai, China

    Huixian Wang, Gaoli Jiang, Dan Dai, Da Hong, Weitao Zhou & Liling Qian

  2. Fujian Provincial Key Laboratory of Neonatal Diseases, Fujian, China

    Liling Qian

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Contributions

(I) Substantial contributions to conception and design: H.W., L.Q.; (II) Acquisition of data, or analysis and interpretation of data: H.W., G.J., D.D., D.H., W.Z., L.Q.; (III) Drafting the article or revising it critically for important intellectual content: H.W., L.Q.; (IV) Final approval of the version to be published: All authors.

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Correspondence to Liling Qian.

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This study was approved by the ethics committees of Children’s Hospital of Fudan University. Written informed consent was obtained from the patient’s guardian.

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Wang, H., Jiang, G., Dai, D. et al. Functional characterization of two novel NKX2-1 frameshift variants that cause pulmonary surfactant dysfunction. Pediatr Res 95, 744–751 (2024). https://doi.org/10.1038/s41390-023-02882-x

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