Environ 10 % des patients atteints de fibrose pulmonaire idiopathique (FPI) ont au moins un apparenté atteint de pneumopathie interstitielle diffuse (PID). Des mutations avaient été mises en évidence sur les gènes codant pour les protéines impliquées dans le métabolisme du surfactant et les protéines du complexe télomérase. Chez l’adulte, les mutations de TERT étaient les plus fréquentes (˜15 %), les mutations de TERC, DKC1 et TINF2 plus rarement retrouvées. Environ 80 % des formes familiales de fibroses pulmonaires chez l’adulte étaient sans cause identifiée. Les objectifs de cette thèse étaient : 1) d’identifier un nouveau gène en cause dans les fibroses pulmonaires familiales de l’adulte inexpliquées, 2) de mieux caractériser le phénotype des patients présentant des mutations de TERT, de TERC ou du nouveau gène mis en évidence.Nous avons sélectionné 35 familles présentant une fibrose pulmonaire familiale pour lesquelles la recherche de mutation TERT, TERC, ABCA3, SFTPB et SFTPC était négative, pour réaliser un séquençage de l’exome. Quatre familles sur les 35 analysées présentaient un variant très rare sur RTEL1 à l’état hétérozygote. La présence des variants a été confirmée par séquençage selon la méthode de Sanger. Ces variants étaient absents des bases de données de contrôles. Les prédictions in silico étaient en faveur du caractère pathogène de ces variants. Les variants co-ségrégeaient avec la maladie dans les familles.L’analyse des variants à partir de la modélisation en 3D de la protéine suggérait un impact fonctionnel des variants sur le site de fixation à l’ATP ou à l’ADN. La taille des télomères des patients étaient raccourcies en comparaison des témoins de la même catégorie d’âge. En 2014, 237 patients avec une fibrose pulmonaire (153 avec une fibrose pulmonaire familiale, 84 avec un syndrome télomère) avaient bénéficié d’un séquençage de TERC et de TERT. Les variants ont été classés comme pathogènes chez 40 patients (16,8 %). Un âge de survenue précoce de fibrose, une macrocytose, ou une thrombopénie étaient significativement associée avec la présence d’une mutation. La probabilité d’une mutation était la plus importante pour les patients de 40-60 ans. La médiane de survie sans transplantation était plus faible pour les patients porteurs de mutations de TERT ou de TERC. Nous avons réalisé un séquençage de l’exome chez 40 autres familles et mis en évidence 5 nouveaux variants de RTEL1 probablement pathogènes in silico. Nous avons par ailleurs mis en évidence 3 autres mutations de RTEL1 dans une cohorte de PID associées à une polyarthrite rhumatoïde. Nous avons colligé les données de 35 patients atteints de PID et porteurs de mutations hétérozygotes de RTEL1. Vingt patients présentaient une FPI (57 %) et 10 une PID secondaire (25,7%). A la différence des mutations de TERT ou TERC, les mutations de RTEL1 étaient associées à moins d’anomalies hématologiques. Par ailleurs, l’expression pulmonaire de la protéine RTEL1 évaluée par immuno-histochimie et de l’ARNm par PCR était équivalente chez les patients porteurs de mutations RTEL1, de TERT ou atteints de FPI sans mutation. Nous avons identifié et confirmé l’implication d’un nouveau gène, RTEL1, dans environ 10 % des fibroses pulmonaires familiales. La présence d’une macrocytose, d’une thrombopénie ou d’un âge jeune en présence d’une forme familiale de fibrose est prédictive de la présence d’une mutation de TERT ou de TERC. La pénétrance des maladies hématologiques semble plus faible pour les mutations de RTEL1 que pour celles de TERT ou TERC dans notre cohorte recrutée sur l’atteinte pulmonaire. Les mutations de TERT ou de RTEL1 sont fréquemment associées à des PID secondaires / About 10% of patients with idiopathic pulmonary fibrosis (IPF) have at least one relative with interstitial lung disease (ILD). Mutations had been reported on the genes encoding for the proteins involved in the surfactant metabolism and in the telomerase complex In adults, TERT mutations were the most frequent (˜15%), mutations of TERC, DKC1 and TINF2 more rarely found. Approximately 80% of the familial forms of pulmonary fibroses in adults were unidentified. The objectives of this work were: 1) to identify a new gene involved in unexplained adult familial pulmonary fibrosis, 2) to better characterize the phenotype of patients with mutations of TERT, TERC or the new gene detected. We selected 35 families with familial pulmonary fibrosis for which the TERT, TERC, ABCA3, SFTPB and SFTPC mutation search was negative, to perform exome sequencing. Four of the 35 families analyzed showed a very rare variant on RTEL1 in the heterozygous state. The presence of the variants was confirmed by Sanger sequencing. These variants were absent from the control databases. In silico predictions were in favor of the pathogenicity of these variants.In families, the variants co-segregated with the disease. In 3D modeling, analysis of the variants suggested a functional impact at the ATP or DNA binding site. The telomere length of carriers of the mutations was shortened compared to controls in the same age group. In 2014, 237 patients with pulmonary fibrosis (153 with familial pulmonary fibrosis, 84 with telomere syndrome) were sequenced for TERC and TERT. The variants were classified as pathogenic in 40 patients (16.8%). An early age of diangosis, macrocytosis, or thrombocytopenia were significantly associated with the presence of a mutation.The probability of a mutation was greatest for patients aged 40-60 years. The median survival without transplantation was lower for patients with TERT or TERC mutations.We performed a sequencing of the exoma in 40 other families and showed 5 new variants of RTEL1 probably pathogenic in silico. We also demonstrated 3 other mutations of RTEL1 in a cohort of ILD associated with rheumatoid arthritis. We collected data from 35 patients with ILD carriers of RTEL1 heterozygous mutations. Twenty patients had IPF (57%) and 10 a secondary ILD (25.7%). Unlike mutations within TERT or TERC, RTEL1 mutations were associated with fewer hematological abnormalities.Furthermore, the pulmonary expression of the RTEL1 protein evaluated by immunohistochemistry and mRNA by PCR was equivalent in patients carriers of RTEL1 or TERT mutations or IPF without mutation. We identified and confirmed the implication of a new gene, RTEL1, in about 10% of familial pulmonary fibroses. The presence of macrocytosis, thrombocytopenia or a young age is predictive of the presence of a mutation within TERT or TERC. The penetrance of hematological diseases appears to be lower for RTEL1 mutation carriers than for TERT or TERC mutation carriers in our cohort of ILD patient. Mutations of TERT or RTEL1 are frequently associated with secondary ILD

RTEL1 (Regulator of telomere length1) DNA helicase has been demonstrated to be vital for the maintenance of telomere length and genomic stability. However, its biological role during development is unknown. Our recent finding that RTEL1 is selectively expressed in several types of adult stem cells, suggests that RTEL1 could play an essential role in the maintenance of these cells. Depending on the function of RTEL1 in the maintenance of genomic stability, we hypothesize that RTEL1 could be required for protecting adult stem cells from genomic instability, whose dysfunction may not only impair tissue homeostasis/regeneration, but also could transform these cells to form tumors. In this study, we have used mouse intestinal stem/progenitor cells model to address this hypothesis. With a transgenic lineage tracing assay, we demonstrated that RTEL1-expressing cells in intestinal crypts can self renew and differentiate to the progeny cells required for intestinal homeostasis. Using a conditional knockout approach, we also showed that loss of RTEL1 function could induce genomic instability in intestinal stem/progenitor cells, which significantly affected the survival of intestinal stem cells and intestinal regeneration. Finally, in this study, we also observed intestinal hyperplasia in our RTEL1 conditional knockout mice, indicating that loss of RTEL1 function may initiate intestinal tumorigenesis. All of these findings strongly support that RTEL1 could be one the key molecules necessary for the maintenance of intestinal stem/progenitor cells and this function could be important for preventing intestinal tumorigenesis.

A fisiopatologia das síndromes de falência da medula óssea (FMO) está relacionada a mecanismos adquiridos de destruição das células-tronco hematopoeiticas na medula ou a defeitos constitucionais em genes fundamentais para o reparo do DNA e manutenção dos telômeros. A anemia aplástica (AA), o protótipo das doenças de FMO, pode ter etiologia adquirida ou constitucional. A avaliação genética de pacientes com AA adquirida tem como objetivo a detecção de mutações somáticas que possam ser usadas como marcadores de resposta ao tratamento imunossupressor. Diferentemente, em pacientes com AA constitucional, a avaliação genética é fundamental para detecção de mutações etiológicas na doença do paciente, sendo essencial para o tratamento e seleção de doadores de medula óssea. Contudo, o papel das mutações constitucionais na fisiopatologia e modulação imunológica da AA adquirida ainda não é conhecido. Neste estudo, nós sequenciamos pacientes com AA de duas coortes independentes utilizando diferentes painéis de sequenciamento de genes alvos. A primeira coorte, composta por 13 pacientes com AA adquirida, foi sequenciada utilizando um painel com 165 genes relacionados à FMO, neoplasias hematológicas, reparo de DNA, manutenção dos telômeros e vias de resposta imune. A segunda coorte, composta por 59 pacientes investigados para doença constitucional, foi sequenciada com um painel de sequenciamento comercial com 49 genes relacionados à FMO hereditária. Foram identificadas alterações potencialmente patogênicas em três dos cinco pacientes com AA adquirida que não responderam à imunossupressão: dois pacientes com variantes em TERT e um com uma variante em DHX36. Não foram identificadas variantes funcionalmente relevantes nos pacientes que responderam ao tratamento imunossupressor. Em contraste, foram identificadas variantes potencialmente patogênicas em RTEL1 em 8 pacientes com AA constitucional. Variantes em RTEL1 foram associadas tanto ao encurtamento telomérico quanto à erosão excessiva do 3\' overhang, independentemente do comprimento dos telômeros. Desse modo, apenas a medida do comprimento dos telômeros não foi suficiente para identificar todos ospacientes com disfunções teloméricas. As plataformas de sequenciamento de nova geração diminuíram o custo e o tempo para a avaliação genética dos pacientes com FMO. Em nosso estudo, os pacientes com AA adquirida não apresentaram um padrão genético associado à sua resposta ao tratamento com imunossupressores, no entanto, o sequenciamento da coorte com suspeita de AA constitucional foi capaz de identificar o defeito genético associado à doença do paciente em 40% dos casos. O uso de dados clínicos, investigação familiar, análises in silico e testes funcionais foram essenciais para uma correta interpretação da patogenicidade de novas variantes identificadas por sequenciamento de nova geração. / The pathophysiology of bone marrow failure (BMF) can be immune, as in acquired aplastic anemia (AA), or constitutional, due to germline mutations in genes critical for DNA repair and telomere maintenance. The genetic screening of patients with constitutional AA is performed to detect germline mutations that are etiologic in patients\' disease. That is critical for treatment decisions and to identify a donor for a bone marrow transplant. In acquired AA, the genetic screening has been used to detect somatic mutations that can predict patients\' outcomes after treatment, as the role of germline mutations in this disease is yet not clear. To investigate the role of germline variants in AA, we screened two independent cohorts with two different targeting sequencing panels; a first cohort composed by 13 patients with acquired AA that was screened using a panel with 165 genes related to BMF, hematologic malignancies, DNA repair, telomere maintenance, and immune response pathways. A second cohort composed of 59 patients suspected to have a constitutional disease screened by a commercial Inherited Bone Marrow Failure Sequencing panel. In our first cohort, while patients without functional relevant germline variants responded to immunosuppression treatment (n=8), three out of 5 nonresponder patients were identified with variants in telomere biology genes. We found patients carrying TERT and DHX36 variants. In our constitutional AA cohort, we identified 8 patients carrying variants in the RTEL1 gene, a helicase critical to telomere maintenance. RTEL1 variants associated with both patients\' overall telomere shortening and single-stranded 3\' overhang erosion independent of telomere length. Also, 3\' overhang erosion was associated with patients\' predisposition to clonal evolution. In this context, the variants identified in the helicases genes DHX36 and RTEL1 were both associated with patients\' normal telomere length and poor outcomes. Also, telomere length measurement alone was insufficient to identify all primary telomere defects. The platforms of next-generation sequencing decreased the cost and time for the genetic screening of patients with BMF. In our study, acquired AA patients did not display a clear genetic pattern associated with their immunosuppressive treatment response. In contrast, the sequencing of the cohort selected based on their suspicion to have an inherited diseaseidentified a molecular defect that might be pathogenic in up to 40% of patients, including the RTEL1 variants. Pathogenicity assessment of genetic variants requires a combination of clinical, in silico, and functional data required to avoid misinterpretation of common variants.

A fisiopatologia das síndromes de falência da medula óssea (FMO) está relacionada a mecanismos adquiridos de destruição das células-tronco hematopoeiticas na medula ou a defeitos constitucionais em genes fundamentais para o reparo do DNA e manutenção dos telômeros. A anemia aplástica (AA), o protótipo das doenças de FMO, pode ter etiologia adquirida ou constitucional. A avaliação genética de pacientes com AA adquirida tem como objetivo a detecção de mutações somáticas que possam ser usadas como marcadores de resposta ao tratamento imunossupressor. Diferentemente, em pacientes com AA constitucional, a avaliação genética é fundamental para detecção de mutações etiológicas na doença do paciente, sendo essencial para o tratamento e seleção de doadores de medula óssea. Contudo, o papel das mutações constitucionais na fisiopatologia e modulação imunológica da AA adquirida ainda não é conhecido. Neste estudo, nós sequenciamos pacientes com AA de duas coortes independentes utilizando diferentes painéis de sequenciamento de genes alvos. A primeira coorte, composta por 13 pacientes com AA adquirida, foi sequenciada utilizando um painel com 165 genes relacionados à FMO, neoplasias hematológicas, reparo de DNA, manutenção dos telômeros e vias de resposta imune. A segunda coorte, composta por 59 pacientes investigados para doença constitucional, foi sequenciada com um painel de sequenciamento comercial com 49 genes relacionados à FMO hereditária. Foram identificadas alterações potencialmente patogênicas em três dos cinco pacientes com AA adquirida que não responderam à imunossupressão: dois pacientes com variantes em TERT e um com uma variante em DHX36. Não foram identificadas variantes funcionalmente relevantes nos pacientes que responderam ao tratamento imunossupressor. Em contraste, foram identificadas variantes potencialmente patogênicas em RTEL1 em 8 pacientes com AA constitucional. Variantes em RTEL1 foram associadas tanto ao encurtamento telomérico quanto à erosão excessiva do 3\' overhang, independentemente do comprimento dos telômeros. Desse modo, apenas a medida do comprimento dos telômeros não foi suficiente para identificar todos ospacientes com disfunções teloméricas. As plataformas de sequenciamento de nova geração diminuíram o custo e o tempo para a avaliação genética dos pacientes com FMO. Em nosso estudo, os pacientes com AA adquirida não apresentaram um padrão genético associado à sua resposta ao tratamento com imunossupressores, no entanto, o sequenciamento da coorte com suspeita de AA constitucional foi capaz de identificar o defeito genético associado à doença do paciente em 40% dos casos. O uso de dados clínicos, investigação familiar, análises in silico e testes funcionais foram essenciais para uma correta interpretação da patogenicidade de novas variantes identificadas por sequenciamento de nova geração. / The pathophysiology of bone marrow failure (BMF) can be immune, as in acquired aplastic anemia (AA), or constitutional, due to germline mutations in genes critical for DNA repair and telomere maintenance. The genetic screening of patients with constitutional AA is performed to detect germline mutations that are etiologic in patients\' disease. That is critical for treatment decisions and to identify a donor for a bone marrow transplant. In acquired AA, the genetic screening has been used to detect somatic mutations that can predict patients\' outcomes after treatment, as the role of germline mutations in this disease is yet not clear. To investigate the role of germline variants in AA, we screened two independent cohorts with two different targeting sequencing panels; a first cohort composed by 13 patients with acquired AA that was screened using a panel with 165 genes related to BMF, hematologic malignancies, DNA repair, telomere maintenance, and immune response pathways. A second cohort composed of 59 patients suspected to have a constitutional disease screened by a commercial Inherited Bone Marrow Failure Sequencing panel. In our first cohort, while patients without functional relevant germline variants responded to immunosuppression treatment (n=8), three out of 5 nonresponder patients were identified with variants in telomere biology genes. We found patients carrying TERT and DHX36 variants. In our constitutional AA cohort, we identified 8 patients carrying variants in the RTEL1 gene, a helicase critical to telomere maintenance. RTEL1 variants associated with both patients\' overall telomere shortening and single-stranded 3\' overhang erosion independent of telomere length. Also, 3\' overhang erosion was associated with patients\' predisposition to clonal evolution. In this context, the variants identified in the helicases genes DHX36 and RTEL1 were both associated with patients\' normal telomere length and poor outcomes. Also, telomere length measurement alone was insufficient to identify all primary telomere defects. The platforms of next-generation sequencing decreased the cost and time for the genetic screening of patients with BMF. In our study, acquired AA patients did not display a clear genetic pattern associated with their immunosuppressive treatment response. In contrast, the sequencing of the cohort selected based on their suspicion to have an inherited diseaseidentified a molecular defect that might be pathogenic in up to 40% of patients, including the RTEL1 variants. Pathogenicity assessment of genetic variants requires a combination of clinical, in silico, and functional data required to avoid misinterpretation of common variants.

Rtel1 est une hélicase qui a été identifiée comme un facteur essentiel pour maintenir les télomères longs et le génome stable chez la souris. Chez l'homme, des mutations germinales dans RTEL1 ont été trouvées chez les patients atteints du syndrome de Hoyeraal-Hreidarsson (HHS), une forme grave de la dyskératose congénitale. Cependant, le mécanisme selon lequel cette protéine agit dans les cellules humaines reste en grande partie inconnu. Pour étudier la fonction de RTEL1 sur le métabolisme des télomères nous avons réduit l'expression de RTEL1 par ARN interférent dans plusieurs lignées de cellules humaines et analysé la longueur des télomères par quantitative-FISH. Nos résultats montrent que la dérégulation de RTEL1 induit un raccourcissement des télomères uniquement dans les cellules avec de très longs télomères et surexprimant la télomérase. Nous démontrons également que l'absence de RTEL1 provoque une altération du complexe de shelterin au télomères: l'augmentation des niveaux de TRF2 et la diminution de POT1. La surexpression de la portion OB fold de POT1 peut restaurer le raccourcissement des télomères causé par le knockdown de RTEL1. Ceci indique que RTEL1 peut jouer un rôle important dans la stabilité du 3' sortant et l'accessibilité de la télomérase. Nous constatons également un impact de RTEL1 sur le métabolisme de l'ARN non codant télomérique TERRA. En effet, la diminution de RTEL1 réduit la quantité totale de TERRA présente dans le noyau et en particulier de TERRA associé aux télomères. Nous constatons que ce nombre réduit de TERRA est causé par sa dégradation, donc nous proposons que RTEL1 a un rôle dans la stabilisation de TERRA aux télomères. / Rtel1, regulator of telomere elongation helicase 1, was discovered as an essential factor for telomere length maintenance and genomic stability in mice. In humans, germline mutations in RTEL1 have been found in patients with Hoyeraal-Hreidarsson syndrome (HHS), a severe form of dyskeratosis congenita. However, the precise mechanism of action of the protein in human cells remains largely unknown. To investigate the function of RTEL1 in human telomere metabolism we used a knockdown approach by specific siRNAs and quantitative-FISH to measure telomere length after depletion of RTEL1 in different cancer cell lines. Our results show that down-regulation of RTEL1 induces shortening of telomeres only in cells with very long telomeres and high telomerase activity. We also demonstrate that upon depletion of RTEL1 there is a different stochiometry of shelterin proteins at telomeres: increased levels of TRF2 and decreased levels of POT1. Importantly, the overexpression of the POT1 OB fold can rescue the shortening of telomeres caused by the knockdown of RTEL1 indicating that RTEL1 may play an important role in the stability of the overhang and in its accessibility to telomerase. We also find an affect of RTEL1 on Telomeric non-coding RNA (TERRA) metabolism. Indeed, depletion of RTEL1 in human cell lines reduces the total amount of TERRA present in the nucleus and in particular of telomere-associated TERRA. Moreover, we find that this reduced number of UUAGGG repeats is caused by TERRA degradation, therefore we propose that RTEL1 has a role in stabilizing TERRA at telomeres.