New Molecular Diagnosis Approaches — From the Identification of Mutations to their Characterization

Molecular diagnosis of cystic fibrosis is based on the detection of mutation in the CFTR gene, identified in 1989. During the past 20 years, thanks to evolutions of diagnostic techniques, our knowledge of mutation spectrum and pathophysiological mechanisms involved in the disease has significantly improved. Sanger sequencing and quantitative methods greatly contributed to the identification of the 2,000 sequence variations reported worldwide in CFTR. We are now entering the new technological age with the generalisation of Next Generation Sequencing (NGS) technologies in diagnostics laboratories. These high throughput approaches allow scanning for the entire CFTR locus, including deep intronic regions, and in parallel other candidate genes that possibly influence the clinical evolution of patients. However, this powerful technology poses new challenge in test interpretation. In this chapter, we review the current and new technologies used in molecular diagnostics of cystic fibrosis, particularly NGS approaches. We also present current and new bioinformatics tools available for the interpretation of variants and in vitro/ex vivo and in vivo techniques that can be used to improve the characterization of the functional impact of CFTR variations

The HDAC inhibitor SAHA does not rescue CFTR membrane expression in Cystic Fibrosis

International audienc

Efficacy of Trastuzumab in Routine Clinical Practice and After Progression for Metastatic Breast Cancer Patients: The Observational Hermine Study

Results of the Hermine study examining the use of trastuzumab for metastatic breast cancer patients in routine practice, including patients who received trastuzumab treatment beyond progression, are reported. The cardiac safety of trastuzumab in this setting is also reported

Epigenetic regulation of CFTR gene

La mucoviscidose (CF) est causée par des mutations sur le gène CFTR codant pour une protéine indispensable au maintien de l'homéostasie des transports hydro-électrolytiques au sein de l'épithélium des organes cibles de la pathologie, dérivés de l'endoderme (poumon, pancréas, appareil reproducteur). Entre ces différents tissus et au cours du développement fœtal, l'expression du gène varie, particulièrement dans le tissu pulmonaire où une répression est observée à l'âge adulte.Ce travail propose dans une première partie la caractérisation des modifications épigénétiques associées à la régulation spatio-temporelle physiologique de l'expression du gène CFTR dans les tissus humains sains adultes et fœtaux. Les résultats obtenus soulignent le rôle important des modifications post-traductionnelles des histones dans la régulation in vivo. Nous avons notamment observé i) un équilibre fin entre marques d'ouverture (acétylation) et de fermeture (H3K27Me3) de la chromatine sur la région promotrice du gène CFTR et ii) l'acétylation significative de régions cis-régulatrices intragéniques.La deuxième partie de ce travail consiste en l'évaluation des effets du SAHA, un inhibiteur d'histones déacétylases (HDACi) dans un modèle ex vivo d'épithélium nasal de patients atteints de mucoviscidose. Les résultats montrent que le SAHA ne restaure pas l'adressage membranaire de la protéine CFTR en contexte pathologique mucoviscidose (mutation p.(Phe508del)) dans des cellules CF différenciées en épithélium ex vivo. De plus, le SAHA induit une modification du profil inflammatoire des épithélia et une dé-différenciation épithéliale dans le modèle ex vivo montrant que les mécanismes d'action de cette molécule sont multiples et réversibles.Ce travail souligne la nécessité d'analyser in vivo les mécanismes physiopathologiques impliqués dans la mucoviscidose et d'évaluer l'impact des molécules thérapeutiques sur les protéines endogènes dans un modèle d'épithélium différencié.Cystic fibrosis (CF) is caused by mutations in the CFTR gene encoding for a protein essential to maintain the homeostasis of fluid and electrolyte transport in the epithelium of endoderm-derived organs (lung, pancreas, reproductive tract) that are affected in CF patients. CFTR expression greatly varies between these tissues and during fetal development, particularly in the lung where repression is observed in adulthood .In the first part of this work, we characterized epigenetic modifications associated with the spatio-temporal regulation of CFTR gene expression in healthy human adult and fetal tissues. Our results emphasize the important role of histone post-translational modifications in this regulation in vivo. Specifically, we observed i) a fine balance between active (acetylation) and repressive (H3K27Me3) marks in the promoter region and ii) significant acetylation in intragenic cis-regulatory regions.In the second part of this work, we evaluated the effects of SAHA, a histone deacetylase inhibitor (HDACi) in an ex vivo model of nasal epithelium of CF patients. Our results show that SAHA can not restore CFTR protein to the apical membrane in a p.(Phe508del)-CFTR context in ex vivo CF differentiated epithelial cells. In addition, SAHA induces a change in the inflammatory profile of epithelia and epithelial dedifferentiation in the ex vivo model showing that the mechanisms of action of this molecule are multiple and reversible.This work highlights the need to analyze in vivo the pathophysiological mechanisms involved in Cystic Fibrosis and to evaluate the impact of therapeutic molecules on the endogenous proteins in a differentiated epithelium model

Epigenetic regulation of CFTR gene

La mucoviscidose (CF) est causée par des mutations sur le gène CFTR codant pour une protéine indispensable au maintien de l'homéostasie des transports hydro-électrolytiques au sein de l'épithélium des organes cibles de la pathologie, dérivés de l'endoderme (poumon, pancréas, appareil reproducteur). Entre ces différents tissus et au cours du développement fœtal, l'expression du gène varie, particulièrement dans le tissu pulmonaire où une répression est observée à l'âge adulte.Ce travail propose dans une première partie la caractérisation des modifications épigénétiques associées à la régulation spatio-temporelle physiologique de l'expression du gène CFTR dans les tissus humains sains adultes et fœtaux. Les résultats obtenus soulignent le rôle important des modifications post-traductionnelles des histones dans la régulation in vivo. Nous avons notamment observé i) un équilibre fin entre marques d'ouverture (acétylation) et de fermeture (H3K27Me3) de la chromatine sur la région promotrice du gène CFTR et ii) l'acétylation significative de régions cis-régulatrices intragéniques.La deuxième partie de ce travail consiste en l'évaluation des effets du SAHA, un inhibiteur d'histones déacétylases (HDACi) dans un modèle ex vivo d'épithélium nasal de patients atteints de mucoviscidose. Les résultats montrent que le SAHA ne restaure pas l'adressage membranaire de la protéine CFTR en contexte pathologique mucoviscidose (mutation p.(Phe508del)) dans des cellules CF différenciées en épithélium ex vivo. De plus, le SAHA induit une modification du profil inflammatoire des épithélia et une dé-différenciation épithéliale dans le modèle ex vivo montrant que les mécanismes d'action de cette molécule sont multiples et réversibles.Ce travail souligne la nécessité d'analyser in vivo les mécanismes physiopathologiques impliqués dans la mucoviscidose et d'évaluer l'impact des molécules thérapeutiques sur les protéines endogènes dans un modèle d'épithélium différencié.Cystic fibrosis (CF) is caused by mutations in the CFTR gene encoding for a protein essential to maintain the homeostasis of fluid and electrolyte transport in the epithelium of endoderm-derived organs (lung, pancreas, reproductive tract) that are affected in CF patients. CFTR expression greatly varies between these tissues and during fetal development, particularly in the lung where repression is observed in adulthood .In the first part of this work, we characterized epigenetic modifications associated with the spatio-temporal regulation of CFTR gene expression in healthy human adult and fetal tissues. Our results emphasize the important role of histone post-translational modifications in this regulation in vivo. Specifically, we observed i) a fine balance between active (acetylation) and repressive (H3K27Me3) marks in the promoter region and ii) significant acetylation in intragenic cis-regulatory regions.In the second part of this work, we evaluated the effects of SAHA, a histone deacetylase inhibitor (HDACi) in an ex vivo model of nasal epithelium of CF patients. Our results show that SAHA can not restore CFTR protein to the apical membrane in a p.(Phe508del)-CFTR context in ex vivo CF differentiated epithelial cells. In addition, SAHA induces a change in the inflammatory profile of epithelia and epithelial dedifferentiation in the ex vivo model showing that the mechanisms of action of this molecule are multiple and reversible.This work highlights the need to analyze in vivo the pathophysiological mechanisms involved in Cystic Fibrosis and to evaluate the impact of therapeutic molecules on the endogenous proteins in a differentiated epithelium model

Inhibiteurs d'histones déacétylases et maladies respiratoires chroniques (effets du SAHA sur un modèle ex vivo d'épithélium nasal de patients atteints de mucoviscidose)

MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

Services publics en réseau : perspectives de concurrence et nouvelles régulations. Un entretien avec Jean Bergougnoux

Marchais-Roubelat Anne, Bergougnoux Jean. Services publics en réseau : perspectives de concurrence et nouvelles régulations. Un entretien avec Jean Bergougnoux . In: Flux, n°41, 2000. pp. 54-63

The Role of Extended CFTR Gene Sequencing in Newborn Screening for Cystic Fibrosis

There has been considerable progress in the implementation of newborn screening (NBS) programs for cystic fibrosis (CF), with DNA analysis being part of an increasing number of strategies. Thanks to advances in genomic sequencing technologies, CFTR-extended genetic analysis (EGA) by sequencing its coding regions has become affordable and has already been included as part of a limited number of core NBS programs, to the benefit of admixed populations. Based on results analysis of existing programs, the values and challenges of EGA are reviewed in the perspective of its implementation on a larger scale. Sensitivity would be increased at best by using EGA as a second tier, but this could be at the expense of positive predictive value, which improves, however, if EGA is applied after testing a variant panel. The increased detection of babies with an inconclusive diagnosis has proved to be a major drawback in programs using EGA. The lack of knowledge on pathogenicity and penetrance associated with numerous variants hinders the introduction of EGA as a second tier, but EGA with filtering for all known CF variants with full penetrance could be a solution. The issue of incomplete knowledge is a real challenge in terms of the implemention of NBS extended to many genetic diseases

Nasal epithelial cells: a tool to study DNA methylation in airway diseases

International audienceA number of chronic airway diseases are characterized by high inflammation and unbalanced activation of the immune response, which lead to tissue damage and progressive reduction of the pulmonary function. Because they are exposed to various environmental stimuli, lung cells are prone to epigenomic changes. Many genes responsible for the immune response and inflammation are tightly regulated by DNA methylation, which suggests that alteration of the epigenome in lung cells may have a considerable impact on the penetrance and/or the severity of airway diseases. A major hurdle in clinical epigenomic studies is to gather appropriate biospecimens. Herein, we show that nasal epithelial cells are suitable to analyze DNA methylation in human diseases primarily affecting the lower airway tract

Reply to Zhongzhong Chen, Hua Xie, and Fang Chen’s Letter to the Editor re: Vuthy Ea, Anne Bergougnoux, Pascal Philibert, et al. How Far Should We Explore Hypospadias? Next-generation Sequencing Applied to a Large Cohort of Hypospadiac Patients. Eur Urol 2021;79:507–15

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