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

Backgroundto 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.Methodspathogenic 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.ResultsThe 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.ConclusionThese results provide novel insights into understanding the molecular mechanism of phenotypes associated with NKX2-1 pathogenic variants

Functional study of NKX2-1 gene mutations associated with lung disease in children

Les anomalies héréditaires du métabolisme du surfactant représentent 10% à 15% des causes de syndromes respiratoires associés à une atteinte pulmonaire alvéolo-interstitielle chez l’enfant. La synthèse de l’ensemble des protéines spécifiques du surfactant (SP-A, SP-B, SP-C et SP-D) et d’ABCA3 est contrôlée par le facteur de transcription NKX2-1 (NK2 homeobox 1). NKX2-1 est un membre de la famille des facteurs de transcription NK-2, identifié initialement comme une protéine nucléaire capable de fixer la thyroglobuline. Son expression est retrouvée au cours du développement du poumon, de la thyroïde et du système nerveux. Chez l’homme, une mutation du gène NKX2-1 a été retrouvée associée à un tableau regroupant de façon parfois incomplète des symptômes neurologiques, thyroïdiens et pulmonaires. Le tableau complet est appelé « Brain-Lung-Thyroid syndrome ». L’atteinte pulmonaire est caractérisée par une pathologie alvéolaire, responsable d’une détresse respiratoire néonatale et/ou d’une pathologie pulmonaire chronique plus ou moins sévère pouvant n’apparaître qu’à l’âge adulte. La triade « Cerveau-Poumon-Thyroïde » est rare et des associations cerveau-thyroïde sans manifestation respiratoire sévère sont le plus souvent décrites. Cette particularité pourrait être liée à un biais de recrutement, ces mutations ayant été recherchées initialement chez des enfants ayant une atteinte thyroïdienne ou neurologique. Nous avons donc initialement décrit l’atteinte pulmonaire, isolée ou non, associée à une mutation de NKX2-1. Cette cohorte a souligné entre autres l’atteinte pulmonaire variée allant de la détresse respiratoire néonatale à la fibrose pulmonaire se manifestant seulement à l’âge adulte, caractéristique des pathologies du surfactant.Notre objectif est donc :1/ d’étudier fonctionnellement les mutations identifiées chez des patients sur les lignées cellulaires pulmonaire et thyroïdienne2/ rechercher des cofacteurs pulmonaires qui pourraient exercer une activité synergique avec NKX2-1 sur les promoteurs des gènes cibles pulmonaires.Dans une première partie, j’ai comparé les mutations NKX2-1DUP et NKX2-1DEL, la première étant responsable de la triade symptomatique et la seconde d’une atteinte pulmonaire isolée. Ces deux mutations qui décalent le cadre de lecture produisent une protéine aberrante de 408 acides aminés avec une perte de l’homéodomaine. La localisation subcellulaire de ces protéines mutées est différente de la protéine sauvage. La protéine NKX2-1DEL est capable de transactiver le promoteur de la thyroglobuline avec le cofacteur PAX8, mais en revanche ne peut activer les promoteurs des gènes spécifiques du surfactant comme la protéine NKX2-1DUP.Dans une deuxième partie, l’analyse d’une famille porteuse de la mutation L191 de NKX2-1 sur trois générations mais dont seul l’enfant présente des symptômes respiratoires, a permis d’identifier par MedExome un variant sur un cofacteur pulmonaire de NKX2-1. L’enfant atteinte est porteuse de ce variant à l’état homozygote contrairement à ces parents et grands-parents. La coexistence de ces 2 variants modifie la localisation subcellulaire de la protéine NKX2-1. L’analyse fonctionnelle a permis de confirmer l’implication de ce deuxième gène dans la pathologie de cette enfant.Ces études contribuent à lever le voile sur l’hétérogénéité clinique des mutations de NKX2-1 et pointent l’importance des cofacteurs tissus spécifiques de la transactivation des gènes cibles pulmonaires et thyroïdiens. De plus, nous décrivons la première variation d’un cofacteur de NKX2-1 ayant un impact fonctionnel pulmonaire. Des mutations de cette protéine pourraient donc être impliquées dans les pathologies interstitielles diffuses isolées de l’enfant.Hereditary abnormalities of surfactant metabolism account for 10% to 15% of the causes of respiratory syndromes associated with interstitial lung disease involvement in children. The synthesis of all surfactant specific proteins (SP-A, SP-B, SP-C and SP-D) and ABCA3 is controlled by the transcription factor NKX2-1 (NK2 homeobox 1). NKX2-1 is a member of the NK-2 transcription factor family, initially identified as a nuclear protein capable of binding thyroglobulin. Its expression is found during the development of the lung, thyroid and nervous system. In humans, a mutation of the NKX2-1 gene has been found associated with neurological, thyroid and/or pulmonary symptoms. The complete picture is called "Brain-Lung-Thyroid Syndrome". The pulmonary involvement is characterized by an alveolar pathology, responsible for a neonatal respiratory distress and / or a more or less severe chronic pulmonary pathology that can only appear in adulthood. The "Brain-Lung-Thyroid" triad is rare and brain-thyroid associations without severe respiratory manifestations are most often described. This particularity could be related to a recruitment bias, these mutations having been sought initially in children having a thyroid or neurological disease. We therefore initially described the pulmonary involvement, isolated or not, associated with a mutation of NKX2-1. This cohort highlighted, among other things, the varied pulmonary involvement ranging from neonatal respiratory distress to pulmonary fibrosis occurring only in adulthood.Our goal is:1 / to functionally study mutations identified in patients on pulmonary and thyroid cell lines2 / to look for pulmonary cofactors that could exert a synergistic activity with NKX2-1 on the promoters of the pulmonary target genes.In a first part, I compared the NKX2-1DUP and NKX2-1DEL mutations, the first being responsible for the symptomatic triad and the second for isolated pulmonary involvement. These two mutations that shift the reading frame produce an aberrant 408 amino acid protein with loss of the homeodomain. The subcellular localization of these mutated proteins is different from the wild-type protein. The NKX2-1DEL protein is capable of transactivating the thyroglobulin promoter with the PAX8 cofactor, but on the other hand cannot activate promoters of the surfactant-specific genes such as the NKX2-1DUP protein.In a second part, the analysis of a family carrying the L191 mutation of NKX2-1 over three generations but of which only the child has respiratory symptoms, allowed to identify by MedExome a variant on a pulmonary cofactor of NKX2. -1. The affected child carries this variant in the homozygous state unlike these parents and grandparents. The coexistence of these 2 variants modifies the subcellular localization of the NKX2-1 protein. Functional analysis has confirmed the involvement of this second gene in the pathology of this child.These studies shed light on the clinical heterogeneity of NKX2-1 mutations and point to the importance of specific tissue cofactors in the transactivation of pulmonary and thyroid target genes. In addition, we describe the first variation of an NKX2-1 cofactor with pulmonary functional impact. Mutations of this protein could therefore be involved in diffuse interstitial pathologies isolated from the child

Methylprednisolone pulse treatment improves ProSP‐C trafficking in twins with <i>SFTPC</i> mutation: An isoform story?

International audienceAims: Mutations in the gene encoding surfactant protein C (SP-C) cause interstitial lung disease (ILD), and glucocorticosteroid (GC) treatment is the most recognized therapy in children. We aimed to decipher the mechanisms behind successful GC treatment in twins carrying a BRICHOS c.566G > A (p.Cys189Tyr) mutation in the SP-C gene (SFTPC). Methods: The twins underwent bronchoscopy before and after GC treatment and immunoblotting analysis of SP-C proprotein (proSP-C) and SP-C mature in bronchoalveolar fluid (BALF). Total RNA was extracted and analysed using quantitative real-time PCR assays. In A549 cells, the processing of mutated protein C189Y was studied by immunofluorescence and immunoblotting after heterologous expression of eukaryotic vectors containing wild type or C189Y mutant cDNA. Results: Before treatment, BALF analysis identified an alteration of the proSP-C maturation process. Functional study of C189Y mutation in alveolar A549 cells showed that pro-SP-C C189Y was retained within the endoplasmic reticulum together with ABCA3. After 5 months of GC treatment with clinical benefit, the BALF analysis showed an improvement of proSP-C processing. SFTPC mRNA analysis in twins revealed a decrease in the expression of total SFTPC mRNA and a change in its splicing, leading to the expression of a second shorter proSP-C isoform. In A549 cells, the processing and the stability of this shorter wild-type proSP-C isoform was similar to that of the longer isoform, but the half-life of the mutated shorter isoform was decreased. Conclusion: These results suggest a direct effect of GC on proSP-C metabolism through reducing the SFTPC mRNA level and favouring the expression of a less stable protein isoform

Deciphering the mechanism of Q145H SFTPC mutation unmasks a splicing defect and explains the severity of the phenotype

International audienceMutations in the gene encoding surfactant protein C (SFTPC) have led to a broad range of phenotypes from neonatal respiratory distress syndrome to adult interstitial lung disease. We previously identified the c.435G4C variant in the SFTPC gene associated with fatal neonatal respiratory distress syndrome in an infant girl. Although this variation is predicted to change glutamine (Q) at position 145 to histidine (H), its position at the last base of exon 4 and the severity of the phenotype suggested that it might also induce a splicing defect. To test this hypothesis, we used hybrid minigene, biochemical and immunofluorescence tools to decipher the molecular mechanism of the mutation. Immunoblotting and confocal imaging showed similar maturation and localization of wild-type and Q145H proteins, but hybrid minigene analysis showed complete exon 4 skipping. Since the exon 4 is in frame, a putative truncated protein of 160 amino acids would be produced. We have shown that this truncated protein had an altered intracellular trafficking and maturation. The c.435G4C mutation is deleterious not because of its amino acid substitution but because of its subsequent splicing defect and should be referred to as r.325_435del and p.Leu109_Gln145del. The absence of residual full-length transcripts fully explained the severity of the phenotype we observed in the infant

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

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

Systematic Severe Acute Respiratory Syndrome Coronavirus 2 Screening at Hospital Admission in Children: A French Prospective Multicenter Study

International audienceTo assess the relevance of systematic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) screening of all children admitted to hospital, we conducted a prospective multicenter study including 438 consecutive hospitalized children. A symptom-based SARS-CoV-2 testing strategy failed to identify 45% (95% confidence interval, 24%-68%) of hospitalized children infected by SARS-CoV-2. To limit intrahospital transmission, a systematic screening of children admitted to hospital should be considered

Heterogeneity of lung disease associated with NK2 homeobox 1 mutations

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