Les genodermatoses sont des maladies génétiques rares à expression cutanée. Parmi elles, les dysplasies ectodermiques (DE) caractérisées par des anomalies du développement d’au moins deux structures ectodermiques (dents, ongles, glandes sudorales et poils), constituent un groupe hétérogène de genodermatoses de plus de 200 syndromes rares. Si la plupart de ces syndromes associent des anomalies des seuls dérivés ectodermiques, d'autres plus complexes, tels que le syndrome de Bazex-Dupré-Christol et l’Incontinentia Pigmenti, rassemblent en plus des manifestations disparates. Le syndrome de Bazex-Dupré-Christol (SBDC) associe une DE à la prédisposition aux carcinomes basocellulaires (CBCs) de survenue précoce. L’étude de 6 familles nous a permis d’identifier, chez 2 d’entre elles, une mutation tronquante dans le gène ACTRT1, codant la protéine Arp-T1. Dans l’épiderme, la protéine Arp-T1 est diminuée chez tous les patients atteints de SBDC, porteurs ou non de mutations dans le gène ACTRT1. Le séquençage à haut débit de la région candidate a permis d’identifier des mutations dans des régions transcrites, régulatrices du gène ACTRT1 chez les patients des 4 autres familles. Notant que la voie Hedgehog est dérégulée dans 70 % des CBCs, nous avons démontré qu’ACTRT1 est un nouvel inhibiteur de cette voie, en particulier par sa liaison au promoteur de GLI1 dont il inhibe l’expression. Enfin, ACTRT1 est un nouveau gène suppresseur de tumeur capable de réduire in vivo la progression tumorale de certaines lignées cancéreuses par la régulation de gènes impliqués dans la prolifération, la mort et la survie cellulaire, ou encore la migration. L’Incontinentia Pigmenti (IP) est une affection multisystémique caractérisée par une atteinte de la peau, des dents, des yeux et parfois du système nerveux central. Elle résulte de mutation dans le gène NEMO et l’abolition de l’activation de la voie NF-KB. L’étude d’une famille concernée par l’IP à permis d’identifier une nouvelle mutation d’épissage du gène NEMO aboutissant à l’expression d’une protéine tronquée. Cette protéine conserve l’intégralité des domaines fonctionnels de NEMO connus à ce jour. Sa caractérisation a révélé une perte d’interaction avec SHARPIN, composants du complexe LUBAC permettant l’ubiquitination linéaire. Il s’agit de la première mutation humaine de NEMO montrant l’importance de son ubiquitination linéaire dans l’activation de la voie NF-KB. Mes travaux de thèse ont ainsi mis en évidence de nouveaux mécanismes physiopathlogiques responsables de deux formes de dysplasie ectodermique. Ces mécanismes reflètent la complexité des voies moléculaires impliquées dans le développement de la peau et le maintien de son homéostasie durant la vie adulte. / Genodermatoses are rare genetic diseases with cutaneous expression. Among them, ectodermal dysplasia (ED) characterized by abnormal development of at least two ectodermal structures (teeth, nails, sweat glands and hair) constitute a heterogeneous group of genodermatoses of more than 200 rare syndromes. While most of these syndromes associate only abnormalities of the ectodermal derivatives, others more complex, such as Bazex-Dupré-Christol syndrome and Incontinentia Pigmenti, bring together disparate manifestations. Bazex-Dupré-Christol syndrome (BDCS) associates ED with predisposition to early basal cell carcinoma (BCCs). The study of 6 families allowed us to identify, in 2 of them, a truncated mutation in the ACTRT1 gene, encoding the Arp-T1 protein. In the epidermis, Arp-T1 protein is decreased in all patients with BDCS, carrying or not of mutations in ACTRT1 gene. High-throughput sequencing of the candidate region allowed to identify mutations in transcribed enhancer regions, regulating the ACTRT1 gene in patients of the remaining 4 families. Noting that the Hedgehog pathway is deregulated in more than 70% of BCCs, we have demonstrated that ACTRT1 is a novel inhibitor of this pathway, via its binding to GLI1 promoter and inhibiting its expression. Finally, ACTRT1 is a new tumor suppressor gene capable of reducing in vivo the tumor progression of certain cancer lines by the regulation of genes involved in proliferation, death and cell survival, or migration. Incontinentia Pigmenti (IP) is a multisystemic disorder characterized by involvement of skin, teeth, eyes and sometimes the central nervous system. It results from mutation in the NEMO gene and the abolition of activation of NF-KB pathway. The study of a family concerned with IP allowed to identify a new splicing mutation of NEMO gene leading to a truncated protein expression. This protein retains all the functional domains of NEMO known. Its characterization revealed a loss of interaction with SHARPIN, components of LUBAC complex allowing linear ubiquitination. This is the first human mutation of NEMO showing the importance of its linear ubiquitination in the activation of the NF-KB pathway. Thus, my thesis work revealed novel physiopathological mechanisms responsible for two forms of ectodermal dysplasia. These mechanisms reflect the complexity of the molecular pathways involved in the development of the skin and the maintenance of its homeostasis during adult life.

<p>Atopic dermatitis (AD) is a common pruritic skin disease in people and domestic animals that can be severely debilitating and stressful to the patient and the caregiver. The diagnosis of AD requires time consuming and expensive procedures, and treatment is often lifelong at considerable cost. Alterations in the lipid composition of the epidermis are a hallmark of the disease, and these may represent changes caused by the inflammation and defects in the lipid barrier. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and, more recently, untargeted profiling using high-resolution time-of-flight instruments have been used to quantify the lipid composition in skin and other tissues, but these approaches are highly demanding in sample preparation and instrument time. In addition, these methods either detect only a limited number of lipids at the time or the identification of detected mass-to-charge ratio (m/z) is problematic when untargeted profiling is used. New lipidomic approaches that generate lipid profiles in a faster and more efficient manner can lead to a better understanding of these lipid changes. </p><p>The mass spectrometry analytical strategy used in this study, multiple reaction monitoring (MRM)-profiling, rapidly identifies discriminant lipids of the epidermis by flow injection. MRM-profiling is a small molecule accelerated discovery workflow performed in two parts using a triple quadrupole mass spectrometer with electrospray ionization as the ion source. Briefly, the first step consists of discovery experiments based on neutral loss and precursor ion scans to detect lipids in pooled samples by targeting class-specific chemical motifs such as polar heads of phospholipids or sphingoid bases of ceramides. The second step of the MRM-profiling is the screening of individual samples for the transitions detected in the discovery phase. </p><p>We first developed the experimental approach of the MRM-profiling methodology using epidermal samples of mice with AD-like inflammatory skin disease (chronic proliferative dermatitis, cpdm). Subsequently, we investigated lipid changes as the disease in mice progressed from minimal to severe. In order to select the most relevant ions, we utilized a two-tiered filter/wrapper feature-selection strategy. First, we built linear models linking the presence of every lipid monitored to disease stage information. The top 10 lipids, ranked based on η2 effect size, were used to build a predictive elastic-net (E-net) regression model linking the lipid ions detected by MRM-profiling with disease progression. The developed model accurately identified disease stages based on the variations in relative amounts of lipid ions corresponding to phosphatidylcholines, cholesterol esters, and glycerolipids-containing and eicosapentaenoic acid fatty acyl residues. Finally, we investigated the lipid profile of the epidermis in dogs with canine AD using the previously developed methodology. Epidermis from client owned patients and healthy controls were collected. Patients were sampled from affected and unaffected skin avoiding areas with secondary infections and the canine atopic dermatitis extent and severity index (CADESI-4) was recorded. The monitored lipids substantially separated the samples of healthy dogs from atopic dogs and distinguished the affected from the unaffected skin of patients. Samples were grouped into two cohorts for low-score and high-score CADESI-4, the first principal component was able to differentiate the control group from the low and high-score group. Differences in the lipid composition associated with low and high score CADESI-4 were significantly different only after separating the samples by sex of the dogs, demonstrating sexual dimorphism in the lipid changes associated with disease. The compositional data was feature extracted using the CADESI-4 to build linear models that identified oleic acid-containing triacylglycerides, long-chain acylcarnitines and sphingolipids as highly predictive lipids and were subsequently used to construct a predictive E-net regression. The lipid fingerprint obtained from the MRM-profiling was highly correlated (R2=0.89) with the classification of the standardized CADESI-4 score. </p><p>This research showed that changes in the lipid composition of the epidermis can be detected by MRM-profiling in atopic dogs even when the skin looks clinically healthy and that sex is a modifying factor in the lipid profile of canine atopic dermatitis (CAD). We expect that this research leads to a better understanding of the lipid changes in the epidermis during the onset of AD and as the chronic inflammatory process develops. The high prediction rate given by the lipid biomarkers for disease progression identified here by the machine learning strategy provides a potential molecular assessment tool for the diagnosis and monitoring of atopic dermatitis and the patient response to treatment.</p><div><br></div>