<p> Mre11 and Rad50 form a complex with Nbs1 (MRN) in mammals and Xrs2 (MRX) in yeast. The MRN complex plays a role in many cellular processes, such as DNA damage sensing, DNA repair, cell cycle checkpoint and telomere maintenance. Rad50 contains a conserved ATP binding motif and its ATPase activity is essential for ATM activation in vitro. Using a tethering approach, I have shown that Rad50 can be targeted to telomeres through its fusion to hRap1. The fusion of hRap1 to Rad50 did not alter the property of Rad50. The fused wild-type Rad50 promoted telomerase-dependent telomere lengthening. However, the fusion proteins containing loss-of-function mutations in Rad50 (K42E and S1202R) did not. I have also shown that the fused wild-type Rad50 was able to form irradiation-induced foci in a manner similar to unfused Rad50. In contrast, the two defective mutants of Rad50 failed to accumulate irradiation-induced foci. Expression of the fusion proteins containing Rad50 mutants also interfered with the ability of endogenous Mre11 protein to form foci post irradiation. Thus our data suggest that the Rad50 mutants may function as dominant-negative alleles in cells.</p> / Thesis / Master of Science (MSc)

Les cellules souches de gliomes (CSG), une sous-population de cellules tumorales, seraient en partie responsables de l’échec des traitements des gliomes de par leur résistance et leur capacité régénérative. Le mécanisme alternatif (ALT) de maintenance des télomères, basé sur la recombinaison homologue et non pas sur la télomérase, est détecté dans environ 30% des gliomes humains suggérant que des stratégies thérapeutiques spécifiquement dirigées contre ALT pourraient avoir un intérêt thérapeutique. Dans ce travail, nous avons poursuivi la caractérisation du premier exemple de CSG humaines ayant un phénotype ALT, les cellules TG20. Nous avons montré que malgré leur très fort taux de recombinaison, les télomères de ces cellules continuaient à assurer leur fonction de protection des chromosomes. Nous avons vérifié que les cellules TG20 conservaient leur capacité à générer des tumeurs intracérébrales après des transplantations sériées chez les souris immunodéprimées tout en gardant un phénotype ALT. Ces résultats confirment à la fois les propriétés de cellules souches cancéreuses des cellules TG20 et la capacité de ALT à assurer la maintenance des télomères nécessaire à l’autorenouvellement et au fort taux de prolifération des CSG in vivo. La greffe intracérébrale de cellules TG20 chez des souris immunodéprimées représente donc un bon modèle d’étude préclinique des gliomes ALT. Nous avons ainsi montré qu’un traitement précoce par un ligand des G-quadruplexes télomériques, 360B, juste après la greffe de cellules TG20, était capable d’inhiber le développement tumoral suggérant l’intérêt de l’utilisation de ligands des G-quadruplexes pour cibler spécifiquement les CSG ALT. Une étude des profils d'expression transcriptomique des cellules TG20 et de plusieurs lignées de CSG humaines exprimant la télomérase, nous a conduit à nous intéresser aux rôles de deux lysine acétyl transférases homologues, PCAF (P300/CBP Associated Factor) et GCN5 (General Control Nonderepressible 5) dans la régulation de la recombinaison télomérique des cellules ALT. Nous avons montré que les inhibitions de ces deux protéines ont des effets opposés sur le mécanisme ALT. Nous proposons qu’une balance d’expression de PCAF et GCN5 régule la maintenance des télomères dans les cellules ALT via le contrôle du turnover de TRF1 ce qui pourrait constituer la base d’une nouvelle stratégie thérapeutique vis-à-vis des gliomes ayant un phénotype ALT. / Glioma stem cells (GSC), a subpopulation of tumor cells, are partly responsible for the failure of treatment of gliomas because of their resistance and regenerative capacity. The mechanism of alternative lengthening of telomere (ALT), based on homologous recombination, is detected in approximately 30 % of human gliomas. Therefore, therapeutic strategies directed specifically against ALT may have a therapeutic value. In this work, we further characterized the first model of human ALT GSC, the TG20 cells. We showed that despite their very high rate of recombination, the telomeres were still capable of fulfilling their protective function of chromosomes. We verified that the TG20 cells retained their ability to generate intracerebral tumors after serial transplantations in immunocompromised mice, while preserving an ALT phenotype. These results confirm the cancer stem properties of TG20 cells and the ability of ALT to ensure telomeres maintenance, which is required for the self-renewal and the high proliferation rate of GSC in vivo. Intracerebral grafts of TG20 cells in immunocompromised mice represent thus a good preclinical model for studying ALT gliomas. We have shown that treatment with a ligand of telomeric G-quadruplexes, the 360B, at an early stage of TG20 tumor engraftment, was able to inhibit tumor growth, showing the interest of the use of G-quadruplex ligands to specifically target ALT GSC. Transcriptomic profiling of TG20 cells and several other GSC telomerase-positive lines, incited us to study the roles of two homologous lysine acetyl transferases, PCAF (p300/CBP Associated Factor) and GCN5 (General Control Nonderepressible 5), in the regulation of telomeric recombination in ALT cells. We showed that the inhibition of these two proteins has opposite effects on the ALT mechanism. We propose that a balance of expression of PCAF and GCN5 regulates the telomere maintenance in ALT cells by controlling the turnover of TRF1. This model could serve for the development of new therapeutic strategies targeting ALT gliomas.

All human chromosomes end with protective structures called telomeres, which consist of thousands of double-stranded TTAGGG repeats and end in a 3’ guanine-rich overhang. These structures shorten normally during each round of replication, and extremely short telomeres along with telomere dysfunction are thought to contribute to the development of aging and cancer. Although many proteins have roles in telomere maintenance, WRN, which is a 3’ to 5’ helicase that is deficient in the premature aging disorder Werner’s syndrome, has been proposed to play multiple roles at telomeres. In this study, I focus on the effect of telomeric sequences and/or structures formed during DNA replication or recombination and how WRN functions at these sites. This study suggests that WRN may promote proper replication of telomeres by accurately aligning telomeric sequences during replication fork regression, potentially the first step in responding to a blockage, such as DNA damage. However, even in the presence of WRN, replication of telomeric sequences is difficult, possibly due to the ability of G-rich sequences to form secondary structures such as G-quadruplexes. I demonstrate that the translesion polymerase pol η, as well as a variety of other polymerases, is unable to synthesize past an intramolecular G-quadruplex formed from telomeric sequence on the template strand. Furthermore, in physiological salt concentrations, WRN favors binding and unwinding a structure that mimics a strand invasion intermediate over other similar structures especially when it possesses G-telomeric sequence. In addition, WRN promotes unwinding of these structures in a direction that would promote additional annealing and strand invasion, supporting a role for WRN in promoting telomeric recombination and formation of a T-loop, a proposed protective structure specific to telomeres. Overall, the data suggest that telomeres may pose problems in replication due to the G-rich, repeating nature of the structures, while WRN may aid in promoting proper replication at these and other replication blocks. Furthermore, WRN may play a role in promoting additional formation of T-loops and other telomeric recombination, thus supporting the relationship of WRN, telomere maintenance, and potentially development of certain aging characteristics.

<p>The centromere is essential for chromosome segregation and genome stability. It is the site of kinetochore assembly and chromosome attachment to the spindle microtubules, and it is important for chromosome movement during mitosis and meiosis. Normal human chromosomes have one centromere, but genome rearrangements that occur with instability, aging, and disease often result in chromosomes with two centromeres, called dicentrics. Nearly seventy-five years ago, Barbara McClintock demonstrated that dicentric chromosomes in plants are associated with instability through mitotic "breakage-fusion-bridge" cycles. However, human dicentrics are unusually stable due to the poorly understood phenomenon of centromere inactivation. Centromere inactivation has been primarily studied in patient-derived dicentrics, limiting the derivation of a molecular pathway. Key centromere and kinetochore proteins are not present at inactive centromeres, but beyond these observations, the process of centromere inactivation is unclear. Epigenetic and sequence-dependent factors are known to contribute to centromere specification, but requirements for centromere assembly, maintenance, and suppression remain obscure. The aims of this research were to (1) determine the mechanism(s) by which de novo dicentric chromosomes are stabilized, (2) ascertain the factors influencing the involvement of specific chromosomes in de novo fusions, and (3) establish the epigenomic, temporal, and mechanistic basis of centromere inactivation. To uncover the mechanistic foundations of these processes, we developed in vitro cell culture systems to study the formation and stabilization of de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. This finding is notable since the most prevalent rearrangement in humans involves the acrocentrics and is called Robertsonian translocation (ROB). In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the centromeric DNA array is reduced compared to the same array before dicentric formation. Many functional dicentrics persist for months after formation. Our results indicate that dicentric human chromosomes undergo alternative fates after formation across a broad temporal window. During transient telomere disruption, we observed a dramatic change in nucleolar appearance. Nucleolar proteins did not coalesce into condensed structures, but appeared dispersed throughout the nucleus. This surprising alteration in nucleolar organization and nuclear architecture suggests remodeling of the nucleolus and subsequent effects on nucleolar-associated chromosomes, such as the acrocentrics, could contribute to the high incidence of ROB formation. Further studies and development of additional cell culture systems will allow us to evaluate current models of centromere assembly and disassembly and the importance of chromatin organization to centromere function and genome architecture.</p> / Dissertation

Les extrémités des chromosomes eucaryotes linéaires ont une structure nucléoprotéique particulière, et sont appelées télomères. Étant donnés leur structure et le mécanisme semi-conservatif de la réplication de l’ADN, la longueur des séquences télomériques est instable. Au fil des divisions cellulaires, les réplications successives de l’ADN entraînent une réduction progressive des séquences télomériques. Des télomères courts ne sont plus fonctionnels, ce qui entraîne l’arrêt du cycle cellulaire et de l’instabilité génomique. Il est donc essentiel de prévenir ce raccourcissement. Une enzyme spécialisée rallonge les télomères : la télomérase. La télomérase est une ribonucléoprotéine (RNP) qui maintient les télomères par un mécanisme d’ajout de répétitions de la séquence télomérique. Afin de former un complexe actif, les sous-unités protéiques de l’enzyme doivent s’assembler autour d’un ARN non-codant, nommé Tlc1 chez la levure Saccharomyces cerevisiae. Cependant, le fait que la RNP nécessite plusieurs sous-unités pour son activité implique un assemblage précis et coordonné. Peu de données existent au sujet de l’assemblage de la RNP en un complexe actif, mais il semble qu’un trafic nucléo-cytoplasmique soit requis dans le cycle fonctionnel de l’enzyme. Caractériser le mécanisme d’assemblage de la télomérase permettra de mieux comprendre les phénomènes de régulation de l’activité de l’enzyme, et donc du maintien des télomères chez S. cerevisiae. À cette fin, j’ai d’abord vérifié l’état stoechiométrique de l’enzyme in vivo par des méthodes de FISH sur des molécules individuelles. J’ai ainsi pu montrer que la télomérase ne comportait qu’un seul ARN Tlc1. Ces données in vivo corrèlent avec des données publiées précédemment grâce à des techniques de biochimie, et suggèrent que l’enzyme n’est composée que de complexes individuels contenant une seule copie de chaque sous-unité protéique. Dans le but d’étudier les mécanismes d’assemblage de la télomérase, j’ai aussi développé un système de contrôle de la transcription d’une forme taguée de Tlc1. Cet outil génétique, basé sur les systèmes Cre-Lox et MS2-GFP, permet l’insertion d’un tag MS2 dans le gène TLC1. Ce tag donne la possibilité de suivre des ARN Tlc1 in vivo et en temps réel par microscopie confocale à spinning-disk. Ce système, baptisé CrEMGaT, a permis de montrer que l’insertion du tag dans le gène entraîne l’apparition de Tlc1-MS2, et que ces ARN forment des agrégats nucléaires ayant des caractéristiques similaires aux T-Recs précédemment caractérisés lors d’une collaboration avec le Pr Chartrand. De plus, des résultats préliminaires obtenus avec le CrEMGaT suggèrent que les ARN Tlc1-MS2 finissent leur cycle fonctionnel au cytoplasme. Dans l’ensemble, les données produites et l’outil développé au cours de cette thèse donnent une meilleure idée de l’état d’assemblage de la télomérase. / Abstract : In the eukaryotic kingdom, the extremities of the linear chromosomes have a particular nucleoproteic structure, and are called telomeres. Because of this structure and the semi-conservative nature of DNA replication, telomere length is unstable. DNA replications during consecutive cell divisions leads to a progressive shortening of telomeric sequences. Below a certain threshold, telomeres are not functional, triggering cell-cycle arrest and genomic instability. It is therefore essential to prevent this shortening. A specialized enzyme elongates telomeres: Telomerase. Telomerase is a ribonucleoprotein (RNP) that adds repeats of the telomeric sequence to the end of telomeres. The enzyme formation requires protein subunits to assemble onto a scaffolding ncRNA, Tlc1 in Saccharomyces cerevisiae. The fact that several subunits are needed for RNP activity implies a precise and coordinated assembly occurs. However, data are lacking about telomerase assembly into an active complex, but different observations point towards a nucleo-cytoplasmic trafficking requirement during the enzyme life-cycle. Characteristics about telomerase assembly mechanism would provide useful information in the quest for understanding the phenomena regulating the enzyme activity, and therefore telomere maintenance in S. cerevisiae. Engaged in this quest, I first verified telomerase stoichiometry in vivo. By quantitative single-molecule FISH, the results showed that the enzyme only contains one Tlc1 RNA per RNP in the cell. These in vivo data correlate with previous publications which, based on biochemical experiments, suggested single copies of the different subunits are present in the complex. Taken together, these findings are dismantling a previous dogma that stipulated telomerase is composed of two complexes, and suggest telomerase quaternary arrangement stays simple. Aiming to study telomerase assembly mechanisms, I also developed an inducible genetic system governing the transcription of a tagged version of Tlc1 (i.e. Tlc1-MS2). This system, based on the Cre-Lox and MS2-GFP systems, allows to control the insertion of a MS2 tag into the TLC1 gene. In this system, dubbed CrEMGaT, the genetic insertion is controllable and indeed leads to Tlc1-MS2 appearance. It is then possible to track these tagged RNAs in vivo and in real time with a spinning disk confocal microscope. Furthermore, these RNAs form nuclear aggregates with characteristics of the T-Recs previously described in a collaboration between our lab and Pr Chartrand’s. Finally, preliminary data obtained with the CrEMGaT suggest cytoplasm is the last cellular compartment visited by Tlc1-MS2 RNA. Overall, these data and the system developed during my thesis will give insights into telomerase assembly in vivo.

Parts of human telomere sequences containing at least 4 guanine subsequences show the ability to form intrastrand quadruplexes of remarkable conformational diversity. Former studies using conventional Raman spectroscopy have revealed that the sequence G3(TTAG3)3 at milimolar concentrations in phosphate buffer solution doped with Na+ ions (ionic strength 150 mM) adopts antiparallel conformation regardless of the length of standing at room temperature or annealing, whereas K+ ions cause gradual transition to "3+1" or even parallel conformation. On the other hand, measurements carried out upon sequence AG3(TTAG3)3 at similar concentrations using Photonic Crystal Fibre-enhanced Raman Spectroscopy (PCFRS) suggest that in the respective presence of both Na+ and K+ (ionic strength 100 mM), a parallel structure is adopted. The hereby presented work employs conventional Raman spectroscopy and Drop Coating Deposition Raman spectroscopy to examine the sequence AG3(TTAG3)3 at concentrations ranging from units to hundreds of milimoles in strands. It concludes that the structure adopted in the presence of Na+, resp. K+ ions is antiparallel, resp. "3+1", and doesn't change over time despite both long standing and annealing. Two hypotheses about the cause of the differences between the results obtained by PCFRS and...

The class Arachnida is not thoroughly explored from the cytogenetic point of view. Previous studies suggest a high diversity of karyotypes and sex determination in arachnids. This study deals with the evolution of sex chomosomes, nucleolar organizer regions (NOR), and telomeric repeats in the tetrapulmonate clade of arachnids, particularly in groups of ancient origin. Sex chromosomes were detected in two orders. Detection of NORs in a large set of species supports the hypothesis that the ancestral karyotype of arachnids contained NOR on one pair of autosomes only. The number of NORs has increased during the evolution of some groups of Pedipalpi. The NORs are located in terminal or subterminal chromosomal regions in most tetrapulmonates. The occurrence of the "insect" telomeric motif was confirmed in majority of tetrapulmonates. Interstitital telomeric repeats were not detected with the exception of one species. Keywords: arachnids, meiosis, sex chromosomes, telomeres, nucleolar organizer, heterochromatin

Dans les cellules somatiques, les télomères s'érodent à chaque division cellulaire. Ce processus appelé « Sénescence Réplicative» est contrebalancé de manière basale chez la levure bourgeonnante S. cerevisiae par l'action de la télomérase qui, alors qu'elle est inactive dans les cellules somatiques des eucaryotes supérieures, est activée dans 85% des cancers. Un autre mécanisme impliqué dans les 15% des cas de cancer restants et est appelé Alternative Lengthening of Telomere (ALT). Dans ce processus, le maintien des télomères est assuré par des mécanismes de recombinaison télomérique induisant des échanges de séquences télomériques de chromatides sœurs (T-SCE).Nous avons évalué l'existence d'ALT dans la LLC-B connue pour rarement exprimer la télomérase. Nous avons montrer que 90% des patients LLC-B présentent une diminution de l'expression de TopoIIIα corrélée à une méthylation plus importante des îlots CpG de la région promotrice du gène suggérant que dans les LLC-B le maintien des télomères est défectueux.Nous avons étudié l'implication de la SUMOylation de TopoIIIα/Top3 dans les mécanismes de régulation du ALT. Nous avons montré que TopoIIIα était SUMOylée in vitro et in vivo au sein des cellules U2-OS ALT. Nous avons aussi observé chez S. cerevisiae que Top3 ne serait SUMOylée qu'en absence d'une activité télomérase. Nos résultats suggèrent que la SUMOylation de TopoIIIα augmenterait son activité in vitro et in vivo en diminuant son affinité pour les télomères une fois la recombinaison achevée et qu'elle serait requise pour son accumulation dans les APBs mais pas pour leur formation. / In somatic cells, telomeres erode with each cell division. This process named « Replicative Senescence » is basically counterbalanced in the budding yeast Saccharomyces cerevisiae by the action of telomerase which, while it is inactive in somatic cells of higher eukaryotes is activated in 85 % of cancer cases. Another process of telomere maintenance is involved in 15% of remaining cancer cases and is called Alternative Lengthening of Telomere (ALT). In this process, telomere maintenance is provided by telomeric recombination mechanisms inducing exchange of telomeric sister chromatid (T-SCE).We assessed the existence of an ALT mechanism in B-CLL known to rarely express telomerase. We have shown that 90% of B-CLL patients have a decreased expression of TopoIIIα correlated with largest methylation of CpG islands of the gene promoter region. Our results suggest that in B-CLL, telomere maintenance is defective either by telomerase or ALT mechanism.We investigated the involvement of post- SUMOylation of TopoIIIα/Top3 in mechanisms regulating ALT phenomenon. We have shown that TopoIIIα was SUMOylated in vitro and in vivo in U2-OS ALT cells. We also observed in S. cerevisiae that Top3p might be SUMOylated in absence of telomerase activity. Our results suggested that the SUMOylation of TopoIIIα increased its activity in vitro and in vivo by reducing its affinity for telomeres once recombination occurred and would be required for its accumulation in APBs but not for their formation.

Thesis advisor: Clare O'Connor / Telomeres are repeated sequences at the ends of chromosomes, which promote chromosome stability by preventing the loss of necessary nucleotides from the DNA with successive rounds of replication. Telomeres are elongated by the enzyme telomerase, which has both a protein component and an RNA component. In the yeast Saccharomyces cerevisiae, the TLC1 gene encodes the RNA component of the enzyme. Telomerase RNA interacts with several proteins to perform its function, including the Ku protein, which binds to the end of the DNA and helps to recruit telomerase to the chromosome thereby facilitating the lengthening of chromosome ends. Ku interacts with telomerase RNA at the site of a 48-nucleotide stem-loop on the RNA's structure. Previous experiments have shown that yeast strains engineered to carry two copies of the TLCI gene exhibit higher levels of telomerase RNA than those that have only one copy of the gene. Also, a yeast strain carrying a copy of the mutant tlc1Δ48 gene, which contains a deletion of the 48-nucleotide stem-loop, contains lower levels of telomerase RNA than a strain with the wild type TLC1 gene. This series of experiments is investigating whether the copy number of the telomerase RNA gene affects the elongation of telomeres in S. cerevisiae. In order to determine this effect, the de novo telomere addition of four strains was examined, as were the native telomere lengths of these strains. The assay indicated that the efficiency of telomere elongation was unchanged by increasing the copy number of the wild type gene but was increased upon increasing the copy number of the mutant gene. Analysis of the native telomere lengths showed that increasing the copy number of either the wild type or the mutant gene allowed the cells to maintain their telomeres at a longer length. / Thesis (BS) — Boston College, 2008. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Pneumonia intersticial familiar (PIF) é definida como a ocorrência de pelo menos dois casos de pneumonia intersticial fibrosante em uma mesma família biológica. Apesar do avanço sobre o tema em anos recentes, ainda não há estudos brasileiros nessa área. O objetivo deste estudo foi caracterizar uma amostra de pacientes brasileiros com PIF quanto aos aspectos clínicos, radiológicos, anatomopatológicos e genéticos, bem como analisar os respectivos comprimentos teloméricos (CT). Entre março de 2014 e novembro de 2017 foi realizada uma busca ativa por casos de PIF. Os pacientes identificados foram submetidos a testes de função pulmonar; tomografia computadorizada de alta resolução (TCAR) de tórax e a coleta de amostras de sangue. Ainda foram realizadas avaliações empregando ficha clínica padronizada. Amostras de tecido pulmonar foram obtidas e revisadas em seis casos. Empregando-se um kit apropriado, DNA genômico foi extraído de células brancas do sangue periférico. A avaliação do CT foi feita pelo método de Southern blot. Um painel envolvendo 154 genes de interesse foi desenvolvido pelo grupo de pesquisa e construído pela empresa Agilent Technologies. A pesquisa desses genes foi realizada empregando-se técnicas de sequenciamento de nova geração (NGS-Illumina). Foram selecionados 35 pacientes, com idade mediana de 66 (35,5-89,3) anos. Tabagismo e outras exposições ambientais estiveram presentes em 45,7% e 80% dos casos, respectivamente. Estertores finos foram identificados em 91,4% dos pacientes e baqueteamento digital em 20%. Os testes de função pulmonar foram classificados como restritivos em 20 (57,1%), normais em 10 (28,6%), inespecíficos em 4 (11,45) e obstrutivo leve em 1 (2,9%). A DCO esteve reduzida nos 30 pacientes em que foi possível pesquisá-la. Padrão típico de PIU na TCAR foi detectado em 6 (17,1%) pacientes e padrão indeterminado para PIU em outros 4 (11,4%). A maioria dos casos, 25 (71,4%), exibiu padrão tomográficoinconsistente com PIU. A revisão do material anatomopatológico revelou pneumonite intersticial com acentuação bronquiolocêntrica em quatro indivíduos. A grande maioria (85,7%) mostrou CTs inferiores ao percentil 50%. Quatro indivíduos exibiram CTs curtos e um muito curto. Foram identificadas variantes genéticas comuns no promotor do gene MUC5B (rs35705950), nos genes TOLLIP (rs111521887, rs5743894 e rs5743890) ou TERT (rs2736100) em 90% dos casos. Detectaram-se ainda sete variantes raras distintas. As alterações c.2594G>A e c.2146G>A do gene TERT, e c.394C>T do gene RTEL1, previamente descritas e associadas a telomeropatias. Uma anormalidade de TERT (c.1730G>A) e outra de RTEL1 (c.2299C>T) inéditas. Duas outras variantes raras encontradas já conhecidas, ainda não haviam sido associadas a doenças pulmonares: gene SHQ1 (c.828_831del) e gene WRAP53 (c.1558dupG). Em conclusão, pacientes brasileiros com PIF demonstram acentuada heterogeneidade fenotípica e genotípica. Este estudo identificou ainda duas novas variantes genéticas raras associadas a PIF e dois possíveis novos genes implicados na patogênese dessa doença. / Familial interstitial pneumonia (FIP) is defined as the occurrence of at least two cases of interstitial fibrosing pneumonias in the same biological family. Despite advances in the field in recent years, there are no Brazilian studies in this area. The aim of this study was to characterize a sample of Brazilian patients with PIF regarding clinical, radiological, histological and genetic aspects, as well as to analyze the respective telomeric lengths (TL). Between March 2014 and November 2017 an active search was conducted for FIP cases. The patients identified were submitted to pulmonary function tests, high resolution computed tomography (HRCT) of the chest and the collection of blood samples. In addition, a standardized clinical file was also fulfilled. Pulmonary tissue samples were obtained and reviewed in six cases. Using a suitable kit, genomic DNA was extracted from white peripheral blood cells. The TL measurements were done by Southern blot method. A panel of 154 genes of interest was developed by the research group and built by Agilent Technologies. Research on these genes was carried out using next generation sequencing techniques (NGSIllumina). Thirty-five patients were selected, with a median age of 66 (35.5-89.3) years. Smoking and other environmental exposures were present in, respectively, 45.7% and 80% of the cases. Fine crackles were identified in 91.4% of patients and digital clubbing in 20%. Pulmonary function tests were classified as restrictive in 20 (57.1%), normal in 10 (28.6%), non-specific in 4 (11.45) and mild obstructive in 1 (2.9%). The DLCO was reduced in the 30 patients in whom it was possible to investigate it. Typical pattern of UIP in HRCT was detected in 6 (17.1%) patients and undetermined pattern for UIP in another 4 (11.4%). The majority of cases, 25 (71.4%), showed a tomographic pattern inconsistent with UIP. The review of histological material revealed interstitial pneumonitis with bronchiolocentric accentuation in four individuals. The vast majority (85.7%) showed TL lower than the 50th percentile. Four individuals had short TL and one a very short TL. Commongenetic variants were identified in the MUC5B gene promoter (rs35705950), in the TOLLIP genes (rs111521887, rs5743894 and rs5743890) or TERT (rs2736100) in 90% of the cases. Seven different rare variants were also detected: the changes c.2594G> A and c.2146G> A of the TERT gene, and c.394C> T of the RTEL1 gene, previously described and associated with telomeropathy; one previously unknown abnormality of TERT (c.1730G> A) and another of RTEL1 (c.2299C> T); two additional rare genetic variants, had not yet been associated with pulmonary diseases: SHQ1 gene (c.828_831del) and WRAP53 gene (c.1558dupG). In conclusion, FIP Brazilian patients demonstrate marked phenotypic and genotypic heterogeneity. This study also identified two new rare genetic variants associated with FIP and two other possible new genes implicated in the pathogenesis of this disease.