Dyskeratosis congenita (DC) is a multi system disorder that exhibits considerable clinical and genetic heterogeneity. It is characterised by mucocutaneous features, bone marrow failure and a predisposition to cancer. Research has identified mutations affecting several telomerase components and patients often have short telomeres, implicating defective telomere maintenance in this disease. Affected components include dyskerin, NOP10 and NHP2, which together with GAR1 form a protein core common to telomerase and all other H/ACA ribonucleoprotein complexes (H/ACA RNPs). Initially characterised as H/ACA RNP components important for pseudouridylation and rRNA processing, their role in the functionally distinct telomerase complex and telomere maintenance is less defined. In order to better understand their implications in DC, this study investigated the importance of these core proteins for the integrity and function of telomerase in human cells. RNAi knockdown studies demonstrated that dyskerin, NOP10 and NHP2 are necessary for the accumulation of TERC (telomerase RNA component); dyskerin and NOP10 for telomerase activity. Moreover, dyskerin was found to be important for maintaining telomere length over time. The impact of NOP10 and NHP2 missense mutations was also analysed in vitro, which indicated that they impair TERC accumulation. The potential effect on pseudouridylation was also considered in this study; the analysis of other H/ACA RNA levels in these knockdown experiments and in a cohort of patients with DKC1 mutations revealed an irregular and inconsistent impact compared to that observed on TERC. Finally, defective telomere maintenance is heavily implicated as the primary cause of DC and very short telomeres have been proposed as a diagnostic marker. This study investigated telomere length in a patient cohort of unprecedented size. It demonstrated the prevalence of the telomere length defect, but telomere length was not found to correlate with either genetic subtype or disease severity, implicating the rate of telomere shortening as the correlating factor instead.

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal the cell to cease division and enter a cell fate termed senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in the telomerase complex or telomere-related genes give rise to the premature aging disorder Dyskeratosis Congenita (DC). DC provides a unique model system to study human aging in relation to telomerase insufficiency and the subsequent accelerated telomere attrition. In this thesis, skin fibroblasts as well as keratinocytes and T-cells were analyzed from patients with Autosomal Dominant Dyskeratosis Congenita (AD DC) caused by a single allele mutation in the telomerase RNA component (TERC) that leads to telomerase haploinsufficiency. These cells were determined to have a severe proliferative defect and extremely short telomeres. It is demonstrated that the short telomeres in AD DC cells initiate a DNA damage response transduced by the p53/p21WAF/CIP pathway which mediate an elevation in steady-state levels of mitochondrially-derived superoxide and oxidative stress. Exogenous expression of the catalytic reverse transcriptase component of telomerase (TERT) activated telomerase in DC fibroblasts but resulted in reduced activity (~50% compared to control fibroblasts); however telomeres were successfully maintained, albeit at a short length. Simultaneous expression of both TERT and TERC led to robust telomerase activity and elongation of telomeres, indicating that TERC haploinsufficiency is a rate-limiting step in telomere maintenance in DC cells. Reconstitution of telomerase activity in AD DC cells ameliorated the proliferative defects, reduced the p53/p21WAF/CIP response and decreased oxidative stress. Increased superoxide and slow proliferation found in DC cells could also be mitigated by inhibiting p21WAF/CIP or by decreasing the oxygen tension to which the cells are exposed. Together, these results support the hypothesis that the insufficient telomerase leads to critically short telomeres which signal the activation of p21WAF/CIP, leading to increased steady-state levels of mitochondrial superoxide and metabolic oxidative stress as a means to engage senescence. These studies provide insight into mechanisms whereby shortened telomeres lead to premature aging in a humans and point to potential strategies to reduce the effects of tissue dysfunction in DC patients.

This thesis details the development of two computational tools, Telomerecat and Parabam, as well as their applications to whole genome sequencing (WGS) data. Telomerecat is a tool for estimating telomere length from WGS data. The strength of Telomerecat lies in its applicability. This applicability is due to a number of advantages over previous attempts to estimate telomere length from WGS. Chief amongst these advantages is that it makes no assumption about the underlying chromosome count or size of the genome within input samples. This means that Telomerecat lends itself well to analysing cancer samples where such assumptions are unfounded. This also means it is applicable to non-human samples, a first for tools of its kind. Furthermore, a novel method for filtering reads derived from interstitial telomere sequences means that it does not rely on previously applied analyses, a source of bias. The other tool described in this thesis is Parabam. Parabam is the first tool of its kind to allow users to apply a function to all of the reads in sequence alignment files, in parallel. Furthermore, Parabam includes a novel method for iterating over index sorted sequence files as if they were name sorted. We provide evidence that Parabam is a quicker way to create complex subsets and statistics from sequence alignment files. In the latter half of the thesis we detail two applications of Telomerecat to large scale WGS projects. The first application, to the Prostate ICGC UK cohort, unveils hitherto uncovered associations between telomere length and previously identified molecular subtypes as well as cancer stage. In the second application, to the NIHR BioResource - Rare Disease cohort, we discover a previously unidentified variant in DKC1 that we propose is directly linked to short telomeres and an immunodeficient phenotype.

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.

Le maintien de l'intégrité du génome est essentiel pour la survie cellulaire et la propagation de l'information génétique. Une mauvaise prise en charge des dommages de l’ADN et / ou une aberration de la maintenance de l’intégrité des télomères - les extrémités des chromosomes linéaires - provoquent chez l'homme des pathologies associées à une instabilité génétique. Ainsi, des dysfonctions télomériques sont à l’origine de la Dyskératose Congénitale (DC), et de sa forme rare et sévère, le Syndrome de Hoyeraal-Hreidarsson (HHS). Les DC et HHS se caractérisent principalement par une insuffisance médullaire progressive, des défauts développementaux et une prédisposition à développer des cancers. Par ailleurs, de nombreux syndromes associant déficits immunitaires et anomalies développementales sont causés par des défauts de réparation de l'ADN (cas de déficits immunitaires sévères, de l’Anémie de Fanconi (FA), de l’ataxie télangiectasie (AT), etc …). Au cours de ce travail, nous avons réalisé une étude phénotypique et génétique de patients atteints de deux pathologies aux caractéristiques cliniques distinctes. Ce travail de thèse a permis : 1) d'une part d'identifier des mutations de RTEL1 chez 3 patients atteints de HHS, décrivant ainsi une nouvelle cause moléculaire de cette pathologie. L'analyse des cellules de ces patients a révélé le rôle crucial que joue RTEL1 sur la stabilité du génome et le maintien des télomères dans des cellules humaines. 2) d'autre part, d'identifier un défaut en MYSM1, une histone déubiquitinase, dans un nouveau syndrome immuno-hématologique associé à des défauts de réparation de l’ADN présentant certaines similitudes avec l'anémie de Fanconi. Cette étude démontre pour la première fois, qu'outre son rôle dans la régulation transcriptionnelle, MYSM1 participe également aux mécanismes de réparation des lésions de l'ADN. / Maintaining genome integrity is essential for cell survival and propagation of the genetic information. Improper management of DNA damages and / or aberrations in maintenance of telomere - the ends of linear chromosomes - causes humans disorders associated with genetic instability. Thus, in humans, telomere dysfunction causes Dyskeratosis Congenita (DC), and its rare and severe form, Hoyeraal-Hreidarsson Syndrome (HHS). DC and HHS are mainly characterized by progressive bone marrow failure, developmental defects and predisposition to cancer. In addition, many syndromes involving immunodeficiency and developmental abnormalities are caused by defects in DNA repair (e.g. severe immune deficiencies, Fanconi Anemia (FA), Ataxia Telangiectasia (AT),…). In this work, we performed a phenotypic and genetic study of patients with two syndromes presenting distinct clinical features. This work permitted : 1) on one hand, to identify RTEL1 mutations in patients with HHS and describe a new molecular cause of this disease. The analysis of patients’ cells revealed the crucial role for RTEL1 in genome stability and telomere maintenance in human cells. 2) on the other hand, to identify mutations in MYSM1, a histone deubiquitinase, in a new immuno-hematological syndrome associated with defects in DNA repair and sharing some similarities with Fanconi anemia. This study demonstrates for the first time that, in addition to its role in transcriptional regulation, MYSM1 is required to cope with DNA damages.