Functional characterization of two novel NKX2-1 frameshift variants that cause pulmonary surfactant dysfunction

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.ImpactPulmonary surfactant dysfunction is a rare heterogeneous disease that exhibits a great burden on children's quality of life.We reported two novel frameshift variants carried by two children with different clinical phenotypes, thus broadening our knowledge base of gene variations and phenotypes in .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 variants.