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

Roles and physiopathological mechanisms of the gene mutations coding the surfactant protein C in the interstitial lung disease development

Les mutations du gène codant pour la protéine C (SP-C) du surfactant pulmonaire (SFTPC) sont à l’origine de pathologies interstitielles chroniques du nourrisson, de l’enfant mais également de l’adulte. Une importante hétérogénéité phénotypique est cependant observée, y compris au sein d’une même famille. Par un épissage alternatif, le gène SFTPC permet la synthèse de deux isoformes du précurseur protéique de SP-C (proSP-C) pour aboutir à la protéine mature après plusieurs modifications post-traductionnelles. Les conséquences des mutations de SFTPC sur l'homéostasie du surfactant ne sont pas clairement élucidées, mais il semble que le mauvais repliement de la protéine soit une caractéristique commune. A l’issue de nos travaux antérieurs, nous avons mis en évidence un effet de certaines mutations et de polymorphismes sur l’épissage de SFTPC faisant ainsi varier significativement l’expression de chacune des deux isoformes protéiques, sans qu’à l’heure actuelle nous ne connaissions le rôle de chacune dans la synthèse de la protéine SP-C mature. Notre projet, s’inscrivant dans la continuité de mon master 2, a pour but de mieux comprendre les mécanismes physiopathologiques pré et post-transcriptionnels associés aux variations de SFTPC et leurs conséquences sur le développement des pneumopathies interstitielles. Le premier axe de notre projet repose sur l’étude in vitro (lignées cellulaires) et in vivo (modèle murin, ARN des patients) des variations de chacun des isoformes. Dans un second axe, nous souhaitons poursuivre l'étude de facteurs pouvant influencer le phénotype des patients porteurs de mutations du gène SFTPC, qu'ils soient d'origine externes (infections virales et bactériennes ou environnementaux comme le tabac) ou génétique. Collectivement, ces études nous permettrons de fournir une signature moléculaire pour cette maladie et d’identifier de nouvelles cibles thérapeutiques afin d’en améliorer le pronostic mais également la prise en charge et la qualité de vie des patients.Surfactant pathologies linked to mutations in the SFTPC gene, via autosomal dominant transmission, are most commonly associated with diffuse interstitial diseases in infants, children and adults, and may also be responsible for acute respiratory distress syndrome in newborns. They are most often accompanied by a high morbidity and mortality rate, thus rendering early diagnosis essential for ideal intervention and support. Mutations in the SFTPC gene lead to alveolar and intracellular accumulation of an abnormal form of the precursor protein SP-C (ProSP-C), which is responsible for the resulting tissue damage. However, the pathophysiological mechanisms are not yet completely deciphered. The gene encodes two isoforms of ProSP-C from three alternative transcripts. The expression level of each is currently unknown and the vast majority of studies evaluating the effect of mutations are performed on only one isoform. Incidentally, our preliminary results on the analysis of RNA extracted from bronchoalveolar washing, both from control subjects and patients harboring a mutation, show that the all three SFTPC transcripts are expressed and that the presence of a mutation is associated with a variation in the expression levels of the transcripts. The aim of my project is to study the expression level of SFTPC transcripts and ProSP-C isoforms from the heterologous expression of the SFTPC gene (exons and introns) in cell lines. I will beanalyzing the post-translational maturation profile of these pro-proteins and evaluating the effect of the mutations on their expression and maturation in both our cellular models and in vivo with two Knock-in mice models.A better understanding of the pathophysiology of genetic abnormalities associated with mutations in the SFTPC gene will not only greatly contribute to earlier management of patients, but also it will help in modifying the progression of lung injury and its prognosis

Long-term outcome of severe paediatric pulmonary Langerhans cell histiocytosis: do not underestimate lung plasticity

International audienceNo abstract availabl

Interstitial lung diseases in children

International audienceInterstitial lung disease (ILD) in children (chILD) is a heterogeneous group of rare respiratory disorders that are mostly chronic and associated with high morbidity and mortality. The pathogenesis of the various chILD is complex and the diseases share common features of inflammatory and fibrotic changes of the lung parenchyma that impair gas exchanges. The etiologies of chILD are numerous. In this review, we chose to classify them as ILD related to exposure/environment insults, ILD related to systemic and immunological diseases, ILD related to primary lung parenchyma dysfunctions and ILD specific to infancy. A growing part of the etiologic spectrum of chILD is being attributed to molecular defects. Currently, the main genetic mutations associated with chILD are identified in the surfactant genes SFTPA1, SFTPA2, SFTPB, SFTPC, ABCA3 and NKX2-1. Other genetic contributors include mutations in MARS, CSF2RA and CSF2RB in pulmonary alveolar proteinosis, and mutations in TMEM173 and COPA in specific auto-inflammatory forms of chILD. However, only few genotype-phenotype correlations could be identified so far. Herein, information is provided about the clinical presentation and the diagnosis approach of chILD. Despite improvements in patient management, the therapeutic strategies are still relying mostly on corticosteroids although specific therapies are emerging. Larger longitudinal cohorts of patients are being gathered through ongoing international collaborations to improve disease knowledge and targeted therapies. Thus, it is expected that children with ILD will be able to reach the adulthood transition in a better conditio

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

Pulmonary Fibrosis in Children

International audiencePulmonary fibrosis (PF) is a very rare condition in children, which may be observed in specific forms of interstitial lung disease. None of the clinical, radiological, or histological descriptions used for PF diagnosis in adult patients, especially in situations of idiopathic PF, can apply to pediatric situations. This observation supports the view that PF expression may differ with age and, most likely, may cover distinct entities. The present review aims at summarizing the current understanding of PF pathophysiology in children and identifying suitable diagnostic criteria. View Full-Tex

Diagnostic workup of childhood interstitial lung disease

Childhood interstitial lung diseases (chILDs) are rare and heterogeneous diseases with significant morbidity and mortality. An accurate and quick aetiological diagnosis may contribute to better management and personalised treatment. On behalf of the European Respiratory Society Clinical Research Collaboration for chILD (ERS CRC chILD-EU), this review summarises the roles of the general paediatrician, paediatric pulmonologists and expert centres in the complex diagnostic workup. Each patient's aetiological chILD diagnosis must be reached without prolonged delays in a stepwise approach from medical history, signs, symptoms, clinical tests and imaging, to advanced genetic analysis and specialised procedures including bronchoalveolar lavage and biopsy, if necessary. Finally, as medical progress is fast, the need to revisit a diagnosis of “undefined chILD” is stressed

Diagnostic workup of childhood interstitial lung disease

Childhood interstitial lung diseases (chILDs) are rare and heterogeneous diseases with significant morbidity and mortality. An accurate and quick aetiological diagnosis may contribute to better management and personalised treatment. On behalf of the European Respiratory Society Clinical Research Collaboration for chILD (ERS CRC chILD-EU), this review summarises the roles of the general paediatrician, paediatric pulmonologists and expert centres in the complex diagnostic workup. Each patient's aetiological chILD diagnosis must be reached without prolonged delays in a stepwise approach from medical history, signs, symptoms, clinical tests and imaging, to advanced genetic analysis and specialised procedures including bronchoalveolar lavage and biopsy, if necessary. Finally, as medical progress is fast, the need to revisit a diagnosis of "undefined chILD" is stressed. Childhood interstitial lung diseases are rare and severe diseases. A stepwise approach to an aetiological diagnosis includes specific investigations performed in expert centres. The term "undefined chILD" must be regularly reassesse