Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2006-2007 / L’anémie de Fanconi (FA) est une maladie génétique caractérisée par une insuffisance médullaire, un risque augmenté de cancers et plusieurs types de malformations. FANCC constitue la seule des 15 protéines FA qui se localise principalement au cytoplasme. De ce fait, en plus de son rôle dans la réparation de l’ADN, FANCC pourrait intervenir dans d’autres mécanismes régulant la croissance des cellules hématopoïétiques. Pour mieux comprendre ses fonctions, nous avons cherché de nouveaux partenaires de FANCC. Le corépresseur de la transcription CtBP1 a été retenu pour analyse. L’étude des interactions a confirmé le lien physique FANCC CtBP1 et révélé que CtBP1 est un membre du complexe FA. Afin d’éclaircir la fonction des interactions, nous avons analysé le profil d’expression génique de cellules réprimées pour les gènes FA ou CtBP1. Les résultats ont montré une expression augmentée de l’antagoniste de la voie Wnt Dickkopf 1 (DKK1). Ainsi, les essais de gènes rapporteurs ont établi que CtBP1 et FANCC agissent comme répresseur transcriptionnel du promoteur DKK1. Nous avons aussi observé que FANCD2 réprime indirectement DKK1 en favorisant l’expression de l’oncogène c Myc. Le mécanisme pourrait dépendre d’interactions entre les protéines FA, incluant FANCC, et les protéines de l’appareil transcriptionnel Wnt, CtBP1 et b caténine. Par ailleurs, nous avons montré que FANCC s’accumule au noyau en réponse à la signalisation Wnt et qu’elle participe avec d’autres protéines FA à l’activation de la b caténine. Ainsi, nous avons trouvé des niveaux élevés de DKK1 dans le surnageant des cellules appauvries en protéines FA et CtBP1, de même que dans les sérums de souris knockout FancA et FancC. Notre étude a permis de montrer que FANCC et les protéines FA, de concert avec CtBP1, agissent dans la régulation transcriptionnelle de l’antagoniste DKK1. Ces résultats suggèrent que FANCC est une protéine clé impliquée dans la signalisation Wnt. Puisque DKK1 est impliqué dans des processus connus pour être perturbés dans la FA, la régulation de DKK1 par FANCC et CtBP1 représente un mécanisme pouvant expliquer la perte progressive des cellules souches hématopoïétiques et représente une étape cruciale pour la découverte de stratégies visant à prévenir l’insuffisance médullaire chez les patients FA. / Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure, excess cancer risk, as well as a broad array of malformations. FANCC is one of fifteen genes linked to the FA disease and encodes a protein that, unlike other FA proteins, is localized primarily to the cell cytoplasm. Because of this, in addition to its role in DNA crosslink repair, FANCC is proposed to function in other mechanisms that can regulate hematopoietic progenitor cell fate. To better understand its functions, we investigated for new partners of the FANCC protein. One candidate, the transcriptional corepressor CtBP1, was selected for further analyses. Interatomic studies confirmed the physical link between FANCC and CtBP1 and revealed that CtBP1 is a member of the FA core complex. To investigate biological function of these interactions, we used a microarray strategy and found that the Wnt antagonist Dickkopf 1 (DKK1) is upregulated in FA and CtBP1 depleted cells. Accordingly, CtBP1 and FANCC were found to act as transcriptional repressor on DKK1 promoter in reporter gene assays. We also observed that FANCD2 indirectly represses DKK1 in promoting the expression of c Myc. Functional mechanism of these repressions may be explained on the observation that FANCC and FA core complex proteins interact with the Wnt transcriptional machinery proteins including CtBP1 and b catenin. Furthermore, we showed that FANCC accumulates into the nucleus in response to Wnt signalisation and participates with other FA proteins in b catenin activation. Therefore, we found increased levels of DKK1 in FA and CtBP1 depleted cells supernatant as well as in sera from FancA and FancC knockout mice. Functional interaction studies showed that FANCC and FA proteins with CtBP1 act in transcriptional regulation of the Wnt antagonist DKK1. These findings suggest that FANCC is a key protein involved in the Wnt signalling response. Because DKK1 is implicated in biological processes similar to those involved in FA pathogenesis, linking FANCC with CtBP1 to the regulation of DKK1 suggests a possible mechanism explaining the progressive loss of bone marrow cells and represents a crucial step for the development of novel strategies aimed at preventing bone marrow failure in FA patients.

Fanconi anemia is an autosomal recessive disorder caused by mutation in one of Fanconi genes and it is manifested by developmental abnormalities, bone marrow failure, predisposition to cancer, cellular sensitivity to cross-linking agents and many other symptoms. Proteins encoded by Fanconi genes and some other proteins are part of Fanconi anemia pathway (FA pathway), which is responsible for DNA repair of an interstrand cross-link (ICL). The repair by this pathway requires monoubiquitination of FANCD2, which is induced and regulated by ATR dependent FANCI phosphorylation. The FANCI phosphorylation initiates the FA pathway but the molecular mechanism of this initialization is not known. Furthermore the proper function of entire pathway requires both: sequence of phosphorylation events of FANCI and monoubiquitination of FANCI:FANCD2 complex . The principle of this work was to study molecular mechanism of initiation and regulation of FA pathway by FANCI phosphorylation. Therefore phosphomimetic mutants of FANCI have been created to investigate their role in processes leading to FANCD2 monoubiquitination. The main aim was to reveal how the phosphorylation of FANCI affects DNA binding and also DNA binding of FANCI:FANCD2 complex. Since both DNA and FANCI phosphorylation are required for proper FANCD2...

Dans le but de reconnaitre et répondre de manière efficace à une très grande variétés d’agents pathogènes, les cellules B ont développé au cours des mécanismes de diversifications des immunoglobulines contrôlés par des processus génétiques complexes comme la recombinaison V(D)J, l’hypermutation somatiques (SHM), et le changement de classe par recombinaison (CSR). L’ensemble de ces processus est contrôlé par différentes voies de réparations de l’ADN. L’anémie de Fanconi est une maladie génétique rare caractérisée par une défaillance progressive de la moelle osseuse, des anomalies de développement et un risque accru de développer des leucémies et des cancers oesopharyngés. La voie FANC est impliquée dans la réparation des pontages de l’ADN et dans le maintien de la fourche de réplication en cas de stress génotoxique. Il est également bien décrit que la voie FANC joue un rôle important dans la coordination des voies de réponses aux dommages à l’ADN. Dans ce travail de thèse, nous nous sommes intéressés au rôle de la voie FANC dans les processus de diversifications des immunoglobulines.En utilisant des souris déficientes pour le gène Fanca, nous montrons que la voie FANC (ou FANCA) participe à la recombinaison V(D)J en contrôlant, dans la moelle osseuse, la transition des cellules B, du stade pre-B au stade de cellules B immatures. Ceci se ferait probablement par le contrôle de la transcription des gènes codant les chaines légères κ des immunoglobulines. Nous montrons également que Fanca pourrait avoir un rôle dans l’addition de nucleotides aux extrémités codantes, en régulant d’une manière indéterminée l’activité et/ou l’activation de l’enzyme TdT ou de la polymérase Polµ. Par ailleurs, nous avons montré que Fanca est nécessaire pour l’induction des mutations de type transitions A/T pendant le processus de SHM en régulant l’expression ou la stabilisation de Polη. Enfin, Fanca (ou la voie FANC) participe à l’inhibition de la recombinaison non homologue (NHEJ) et est requis durant le CSR pour stabiliser les duplexes entre 2 régions de microhomologies qui facilitent le recrutement d’endonucléases et réguler l’accès des DNA polymérases aux cassures de l’ADN. / To recognize and respond dynamically to an enormous variety of different pathogens, B lymphocytes of the immune system have evolved controlled genetic processes at their immunoglobulin (Ig) loci that are known as Ig diversification including V(D)J recombination, somatic hypermutation (SHM), and class switch recombination (CSR). These complex and vulnerable processes are orchestrated by multiple DNA repair pathways. Fanconi anemia (FA) is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and an increased risk of leukemia and cancer. FANC pathway has been implicated in DNA interstrands crosslinks (ICL) repair and in the rescue of stalled replication forks. The FANC pathway also plays a fundamental role in coordinating the DNA repair pathways. Several lines of evidence suggest a possible involvement of the FANC pathway in Ig diversification processes, thus we are particularly interested in revealing function of FANC pathway during these processes. By using Fanca-/- mice, our results first show that during V(D)J recombination, Fanca (or FANC pathway) participates to the control of the transition from pre-B to immature B cells in bone marrow (BM), probably through transcriptional activation of post-rearranged κ light chain. In addition, Fanca might play a role in nucleotide addition at coding end, possibly by regulating either TdT or Polµ activity/activation. Secondly, we found that Fanca is required for the induction of transition mutations at A/T during SHM via regulation of Polη expression/stabilization. Finally, Fanca (or FANC pathway) inhibits short-range recombination and is required during CSR to stabilize duplexes between 2 short microhomology regions that facilitate the recruitment of endonucleases to trim overhangs and avoid unscheduled access of polymerases to DNA ends.

Cette thèse est consacrée à la modélisation qualitative des réseaux biologiques pour l'innovation thérapeutique. Elle étudie comment utiliser les réseaux Booléens, et comment les améliorer, afin d'identifier des cibles thérapeutiques au moyen d'approches in silico. Elle se compose de deux travaux : i) un algorithme exploitant les attracteurs des réseaux Booléens pour l'identification in silico de cibles dans des modèles Booléens de réseaux biologiques pathologiquement perturbés, et ii) une amélioration des réseaux Booléens dans leur capacité à modéliser la dynamique des réseaux biologiques grâce à l'utilisation des opérateurs de la logique floue et grâce au réglage des arrêtes. L'identification de cibles constitue l'une des étapes de la découverte de nouveaux médicaments et a pour but d'identifier des biomolécules dont la fonction devrait être thérapeutiquement modifiée afin de lutter contre la pathologie considérée. Le premier travail de cette thèse propose un algorithme pour l'identification in silico de cibles par l'exploitation des attracteurs des réseaux Booléens. Il suppose que les attracteurs des systèmes dynamiques, tel que les réseaux Booléens, correspondent aux phénotypes produits par le système biologique modélisé. Sous cette hypothèse, et étant donné un réseau Booléen modélisant une physiopathologie, l'algorithme identifie des combinaisons de cibles capables de supprimer les attracteurs associés aux phénotypes pathologiques. L'algorithme est testé sur un modèle Booléen du cycle cellulaire arborant une inactivation constitutive de la protéine du rétinoblastome, tel que constaté dans de nombreux cancers, tandis que ses applications sont illustrées sur un modèle Booléen de l'anémie de Fanconi. Les résultats montrent que l'algorithme est à même de retourner des combinaisons de cibles capables de supprimer les attracteurs associés aux phénotypes pathologiques, et donc qu'il réussit l'identification in silico de cibles proposée. En revanche, comme tout résultat in silico, il y a un pont à franchir entre théorie et pratique, requérant ainsi une utilisation conjointe d'approches expérimentales. Toutefois, il est escompté que l'algorithme présente un intérêt pour l'identification de cibles, notamment par l'exploitation du faible coût des approches computationnelles, ainsi que de leur pouvoir prédictif, afin d'optimiser l'efficience d'expérimentations coûteuses. La modélisation quantitative en biologie systémique peut s'avérer difficile en raison de la rareté des détails quantitatifs concernant les phénomènes biologiques, particulièrement à l'échelle subcellulaire, l'échelle où les médicaments interagissent avec leurs cibles. Une alternative permettant de contourner cette difficulté est la modélisation qualitative étant donné que celle-ci ne requiert que peu ou pas d'informations quantitatives. Parmi les méthodes de modélisation qualitative, les réseaux Booléens en sont l'une des plus populaires. Cependant, les modèles Booléens autorisent leurs variables à n'être évaluées qu'à vrai ou faux, ce qui peut apparaître trop simpliste lorsque des processus biologiques sont modélisés. En conséquence, le second travail de cette thèse propose une méthode de modélisation dérivée des réseaux Booléens où les opérateurs de la logique floue sont utilisés et où les arrêtes peuvent être réglées. Les opérateurs de la logique floue permettent aux variables d'être continues, et ainsi d'être plus finement évaluées qu'avec des méthodes de modélisation discrètes tel que les réseaux Booléens, tout en demeurant qualitatives. De plus, dans le but de considérer le fait que certaines interactions peuvent être plus lentes et/ou plus faibles que d'autres, l'état des arrêtes est calculé afin de moduler en vitesse et en force le signal qu'elles véhiculent. La méthode proposée est illustrée par son implémentation sur un petit échantillon de la signalisation du récepteur au facteur de croissance épidermique... [etc] / This thesis is devoted to the qualitative modeling of biological networks for therapeutic innovation. It investigates how to use the Boolean network formalism, and how to enhance it, for identifying therapeutic targets through in silico approaches. It is composed of two works: i) an algorithm using Boolean network attractors for in silico target identification in Boolean models of pathologically disturbed biological networks, and ii) an enhancement of the Boolean network formalism in modeling the dynamics of biological networks through the incorporation of fuzzy operators and edge tuning. Target identification, one of the steps of drug discovery, aims at identifying biomolecules whose function should be therapeutically altered in order to cure the considered pathology. The first work of this thesis proposes an algorithm for in silico target identification using Boolean network attractors. It assumes that attractors of dynamical systems, such as Boolean networks, correspond to phenotypes produced by the modeled biological system. Under this assumption, and given a Boolean network modeling a pathophysiology, the algorithm identifies target combinations able to remove attractors associated with pathological phenotypes. It is tested on a Boolean model of the mammalian cell cycle bearing a constitutive inactivation of the retinoblastoma protein, as seen in cancers, and its applications are illustrated on a Boolean model of Fanconi anemia. The results show that the algorithm returns target combinations able to remove attractors associated with pathological phenotypes and then succeeds in performing the proposed in silico target identification. However, as with any in silico evidence, there is a bridge to cross between theory and practice, thus requiring it to be used in combination with wet lab experiments. Nevertheless, it is expected that the algorithm is of interest for target identification, notably by exploiting the inexpensiveness and predictive power of computational approaches to optimize the efficiency of costly wet lab experiments. Quantitative modeling in systems biology can be difficult due to the scarcity of quantitative details about biological phenomenons, especially at the subcellular scale, the scale where drugs interact with there targets. An alternative to escape this difficulty is qualitative modeling since it requires few to no quantitative information. Among the qualitative modeling approaches, the Boolean network formalism is one of the most popular. However, Boolean models allow variables to be valued at only true or false, which can appear too simplistic when modeling biological processes. Consequently, the second work of this thesis proposes a modeling approach derived from Boolean networks where fuzzy operators are used and where edges are tuned. Fuzzy operators allow variables to be continuous and then to be more finely valued than with discrete modeling approaches, such as Boolean networks, while remaining qualitative. Moreover, to consider that some interactions are slower and/or weaker relative to other ones, edge states are computed in order to modulate in speed and strength the signal they convey. The proposed formalism is illustrated through its implementation on a tiny sample of the epidermal growth factor receptor signaling pathway. The obtained simulations show that continuous results are produced, thus allowing finer analysis, and that modulating the signal conveyed by the edges allows their tuning according to knowledge about the modeled interactions, thus incorporating more knowledge. The proposed modeling approach is expected to bring enhancements in the ability of qualitative models to simulate the dynamics of biological networks while not requiring quantitative information. The main prospect of this thesis is to use the proposed enhancement of Boolean networks to build a version of the algorithm based on continuous dynamical systems...[etc]

The DNA damage response (DDR) pathway is a complicated network of interacting proteins that function to sense and remove DNA damage. Upon exposure to DNA damage, a signaling cascade is generated. The damage is either removed, restoring the original genetic sequence, or apoptosis is activated. In the absence of DDR, cells are unable to effectively process DNA damage. Unprocessed DNA damage can lead to chromosomal changes, gene mutations, and malignant transformation. Thus, the proteins involved in DDR are critical for maintaining genomic stability. One essential DDR protein is the BRCA1 Associated C-terminal Helicase, BACH1. BACH1 was initially identified through its direct association with the BRCT domain of the Breast Cancer Associated Gene, BRCA1. Similar to BRCA1, germline mutations in BACH1were identified in patients with early onset breast cancer. Interestingly, the disease-associated mutations in BACH1 were shown to have altered helicase activity in vitro, providing a direct link between BACH1 helicase activity and disease development. The correlation between BACH1 and cancer predisposition was further confirmed by the identification of BACH1 as the cancer syndrome Fanconi anemia (FA) gene product, FANCJ. Similar to other FA proteins, suppression of FANCJ leads to decreased homologous recombination, enhanced sensitivity to DNA interstrand crosslinking (ICL) agents, and chromosomal instability. In an effort to further understand the function of FANCJ in DDR, FANCJ was shown to directly associate with the mismatch repair (MMR) protein MLH1. This interaction is facilitated by lysines 141 and 142 within the helicase domain of FANCJ. Importantly, the FANCJ/MLH1 interaction is critical for ICL repair. Furthermore, in an attempt to dissect the binding site of FANCJ on MLH1, we discovered an HNPCC associated MLH1 mutation (L607H) that has intact mismatch repair, but lacks FANCJ interaction. In contrast to the MLH1 interaction, the FANCJ/BRCA1 interaction was not required for correcting the cellular defects in FANCJ null cells. Thus, in an effort to understand the functional significance of the FANCJ/BRCA1 interaction, we discovered that FANCJ promotes Pol η dependent translesion synthesis (TLS) bypass when uncoupled from BRCA1. In this thesis, we provide evidence suggesting that FANCJ and MLH1 are functionally linked and that the interaction of these proteins is critical for repair choice.

Telomeres are the chromosome end structures consisting of telomere-associated proteins and short tandem repeat sequences, TTAGGG, in humans and mice. Telomeres prevent chromosome termini from being recognized as broken DNA ends. The structural integrity of DNA including telomeres is constantly threatened by a variety of DNA damaging agents on a daily basis. To counteract the constant threats from DNA damage, organisms have developed a number of DNA repair pathways to ensure that the integrity of genome remains intact. A number of DNA repair proteins localize to telomeres and contribute to telomere maintenance; however, it is still unclear as to what extent. Telomere shortening has been linked to rare human disorders that present with bone marrow failure including Fanconi anemia (FA). FANCC is one of the most commonly mutated FA genes in FA patients and the FANCC subtype tends to have a relatively early onset of bone marrow failure and hematologic malignancies. Here, we studied the role of Fancc in telomere length regulation in mice. We demonstrated that deletion of Fancc did not affect telomerase activity, telomere length or telomeric end-capping in mice with long telomeres. We also showed that Fancc deficiency accelerates telomere shortening during high turnover of hematopoietic cells and promotes telomere recombination initiated by short telomeres. Telomere shortening has also been linked to human aging and cancer development, with oxidative stress as a major contributing factor. 8-oxo-7, 8-dihydroguanine is among the most common oxidative DNA lesions, and is substrates for OGG1-initiated DNA base excision repair. Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity. We demonstrated that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. We also examined if telomeric DNA is particularly susceptible to oxidative guanine damage and if telomere specific factors affect the incision of oxidized guanines by OGG1. We showed that the GGG sequence context of telomere repeats and certain telomere configurations may contribute to telomere vulnerability to oxidative DNA damage processing.

Per aprofundir en les causes genètiques de l’anèmia de Fanconi (FA) i la susceptibilitat al càncer de mama (BrCa), així com en els mecanismes moleculars de reparació del dany al DNA per part de la via de senyalització FA/BrCa, s’ha dut a terme un cribatge massiu d’interaccions proteïna-proteïna per 34 components involucrats en aquesta via. Aquest estudi ha identificat 25 noves interaccions d’alta fiabilitat, la majoria de les quals estan involucrades en la regulació del cicle cel•lular o la resposta al dany al DNA. Algunes d’aquestes interaccions han estat validades mitjançant estudis de co-immunoprecipitació endògena i sobreexpressió en cèl•lules de mamífer. En aquesta tesi doctoral hem caracteritzat la nova interacció entre FANCN/PALB2 ¡ MORF4L1/MRG15. S’han realitzat estudis bioquímics i funcionals amb la finalitat de caracteritzar-la tan molecularment com funcionalment en cèl•lules d’humans, de ratolí i Caenorhabditis elegans. Els resultats obtinguts ens permeten dibuixar un possible mòdul funcional entre BRCA2, PALB2, RAD51 i RPA1 que suggereixen una implicació de MRG15 en la reparació dels trencaments de doble cadena (DSB) en el DNA. Els nostres resultats no mostren alteracions patogèniques de MORF4L1/MRG15 en els pacients FA amb mutació desconeguda. Pel que fa a l’estudi de dos variants genètiques comunes en el locus MORF4L1, tampoc s’observa una associació significativa amb el risc a càncer de mama entre els portadors de mutació BRCA1 i BRCA2; tot i que s’observa una tendència entre els portadors de mutacions BRCA2. És per aquest motiu, que serà necessari un anàlisi amb sèries de portadors més grans. / Towards a full model of the molecular network altered in Fanconi anemia and breast cancer Proteins encoded by Fanconi anemia (FA) and/or breast cancer (BrCa) susceptibility genes cooperate in a common DNA damage repair signaling pathway. There are fiveteen FA complementation groups (FANCs) that encode for proteins of this pathway and three of these genes overlap between FA and BrCa. With the objective to gain deeper insight into this pathway and its influence on cancer risk, we search for novel components through protein physical interaction screens. Protein physical interactions were screened using the yeast two-hybrid system (Y2H). Novel interactions were validated through co-immunoprecipitation and co-affinity purification assays. A previously identified co-purifying protein with PALB2 was indentified MRG15 (MORF4L4 gene). Biochemical and functional assays were performed to characterize the molecular interaction in human, mouse and Caenorhabditis elegans. Results obtanined draw molecular and functional relationships with BRCA2, RAD51 and RPA that suggest a role for MRG15 in the repair of DNA double-strand breaks. Mrg15 deficient murine embryonic fibroblast showed intermediate sensitivity to γ-radiation relative to controls and impaired Rad51 foci formation. No alterations or mutations were identified for MORF4L1 in unclassified FA patients and BrCa familial cases. However, a potential modifier effect for a common genetic variation in MORF4L1 can not be ruled out among BRCA2 mutation carriers.

Student Number : 9807768F - MSc (Med) research report - Faculty of Health Sciences / Fanconi anaemia (FA) is an autosomal recessive disorder characterised by aplastic anaemia (AA) and a high risk of developing acute myeloid leukaemia (AML). It is unknown whether heterozygote carriers are also predisposed to developing these disorders. The black South African population group is ideal for FA mutation screening because the presence of a founder mutation, FANCG 637-643, makes screening relatively straight forward. Three individuals with AML (115 screened) and one with AA (78 screened) were found to be heterozygous for the black South African founder mutation. From our data it seems unlikely that this mutation places heterozygous carriers of the mutation at high risk of developing AML or AA. Three children with AA out of 26 screened, were homozygous for the mutation. This finding reiterates the importance of screening all children with AA for FA. The frequency of certain congenital abnormalities in black South African FA patients was compared to patients described by other research groups. The frequencies of the abnormalities were similar to other FANCG cohorts described but significant differences to a group of FA patients from unspecified complementation groups were found. This difference could be because different complementation groups are associated more or less strongly with specific abnormalities. It was found previously that particular congenital abnormalities in FA patients are associated with a poor haematological outcome. We concluded that black South African FANCG patients have a high risk of early development of AA even though they do not have a high frequency of congenital abnormalities.

A Anemia de Fanconi (AF) é uma doença genética rara de herança recessiva caracterizada por insuficiência da medula óssea, anormalidades de desenvolvimento malformações congênitas. A doença evolui para anemia aplástica grave em decorrência da falência progressiva da medula óssea e neoplasias malignas, principalmente a leucemia mielóide aguda e os carcinomas espinocelulares em região de cabeça e pescoço. O risco de desenvolvimento de neoplasias malignas aumenta após o transplante de medula óssea TMO), e recentemente foi proposta a participação do papilomavírus humano (HPV) na carcinogênese na AF. Neste estudo foram realizados exame físico da boca, coleta de amostras de esfregaços de células epiteliais orais por meio da citologia em base líquida, avaliação quanto à presença de atipias de acordo com a classificação de Papanicolaou, análise citomorfométrica das células epiteliais orais, detecção do HPV por meio de técnicas de PCR e tipificação das amostras positivas por meio de hibridização reversa. A amostra foi dividida em 4 grupos: pacientes com AF transplantados (I) e não transplantados de medula óssea (II), pacientes com Anemia aplástica severa (AAS) transplantados (III) e um grupo de indivíduos saudáveis (controle) (IV). Ao exame físico não foram detectadas lesões malignas. Os resultados mostraram que, em comparação ao grupo controle: a) houve aumento da área do núcleo (NA) das células epiteliais orais na AF e na AAS (p<0,05); b) houve aumento da área do citoplasma (CA) na AF (p<0,05); c) houve diminuição da razão NA/CA na AF em pacientes transplantados (p<0,05), aumento desta na AAS (p<0,05) e ausência de alterações nos pacientes com AF não transplantados de medula óssea; d) foi encontrada alta prevalência do HPV nos pacientes tranplantantados de medula óssea para AF (35%, p<0,05) e AAS (38%, p<0,05), enquanto que nos pacientes com AF não transplantados 27,27%) não houve diferença estatisticamente significativa em relação ao grupo controle 6,25%) e e) o HPV-16, de alto risco para transformação maligna, foi o genótipo mais freqüente (grupo I 71,42%, grupo II 33,33%, grupo III 28,57%, grupo IV 0%). O risco relativo em apresentar a infecção pelo HPV foi maior nos pacientes transplantados, tendo sido influenciado pela idade e pelo tempo de TMO. Em conclusão, estes resultados demonstraram que existem alterações na morfometria das células epiteliais orais na AF e há alta prevalência de HPVs, especialmente HPV-16, na boca de pacientes sem lesões orais malignas. / Fanconis anemia (FA) is a rare recessive genetic disorder characterized by boné marrow failure, developmental malformations and congenital abnormalities. FA progressive bone marrow failure usually evolves to aplastic anemia and malignant neoplasias, especially acute myeloid leukemia and head and neck squamous cell carcinomas. The risk of malignancies is increased after bone marrow transplantation (BMT), and recently the role of human papillomavirus (HPV) in FA carcinogenesis was proposed. We performed an oral examination, liquid based cytology sampling of oral epithelial cells, assessment of atypias according to Papanicolaou classification, cytomorphometric analysis of oral epithelial cells, HPV screening by PCR and genotyping by reverse hybridization. Sample subjects were divided into 4 groups: BMT FA patients (I), not BMT FA patients (II), BMT severe aplastic anemia patients (SAA) and a healthy control group (IV). No malignant lesions were detected on oral examination. In comparison with the control group the results showed: a) an increase in the nuclear area (NA) in oral epithelial cells in FA and SAA (p<0.05); b) an increase in the cytoplasmic area (CA) in FA (p<0.05); c) a decrease in the NA/CA ratio in BMT FA patients (p<0.05), an increase of it in SAA (p<0.05) and no alterations in not BMT FA patients; d) a high HPV prevalence was detected in BMT FA (35%, p<0.05) and SAA patients (38%, p<0.05), whereas in not BMT FA patients (27.27%) there was not a significant statistical difference in comparison with the control group (6.25%) and e) the high risk, HPV-16, was the most frequent genotype (group I 71.42%, group II 33.33%, group III 28.57%, group IV 0%). HPV infection odds ratio adjusted for age and years of BMT showed an increase in HPV infection for both factors. In conclusion, results demonstrated that there were morphometric alterations in oral epithelial cells in FA and a high prevalence of oral HPV, especially HPV-16, in these patients without malignant lesions.

A Anemia de Fanconi (AF) é uma doença genética rara de herança recessiva caracterizada por insuficiência da medula óssea, anormalidades de desenvolvimento malformações congênitas. A doença evolui para anemia aplástica grave em decorrência da falência progressiva da medula óssea e neoplasias malignas, principalmente a leucemia mielóide aguda e os carcinomas espinocelulares em região de cabeça e pescoço. O risco de desenvolvimento de neoplasias malignas aumenta após o transplante de medula óssea TMO), e recentemente foi proposta a participação do papilomavírus humano (HPV) na carcinogênese na AF. Neste estudo foram realizados exame físico da boca, coleta de amostras de esfregaços de células epiteliais orais por meio da citologia em base líquida, avaliação quanto à presença de atipias de acordo com a classificação de Papanicolaou, análise citomorfométrica das células epiteliais orais, detecção do HPV por meio de técnicas de PCR e tipificação das amostras positivas por meio de hibridização reversa. A amostra foi dividida em 4 grupos: pacientes com AF transplantados (I) e não transplantados de medula óssea (II), pacientes com Anemia aplástica severa (AAS) transplantados (III) e um grupo de indivíduos saudáveis (controle) (IV). Ao exame físico não foram detectadas lesões malignas. Os resultados mostraram que, em comparação ao grupo controle: a) houve aumento da área do núcleo (NA) das células epiteliais orais na AF e na AAS (p<0,05); b) houve aumento da área do citoplasma (CA) na AF (p<0,05); c) houve diminuição da razão NA/CA na AF em pacientes transplantados (p<0,05), aumento desta na AAS (p<0,05) e ausência de alterações nos pacientes com AF não transplantados de medula óssea; d) foi encontrada alta prevalência do HPV nos pacientes tranplantantados de medula óssea para AF (35%, p<0,05) e AAS (38%, p<0,05), enquanto que nos pacientes com AF não transplantados 27,27%) não houve diferença estatisticamente significativa em relação ao grupo controle 6,25%) e e) o HPV-16, de alto risco para transformação maligna, foi o genótipo mais freqüente (grupo I 71,42%, grupo II 33,33%, grupo III 28,57%, grupo IV 0%). O risco relativo em apresentar a infecção pelo HPV foi maior nos pacientes transplantados, tendo sido influenciado pela idade e pelo tempo de TMO. Em conclusão, estes resultados demonstraram que existem alterações na morfometria das células epiteliais orais na AF e há alta prevalência de HPVs, especialmente HPV-16, na boca de pacientes sem lesões orais malignas. / Fanconis anemia (FA) is a rare recessive genetic disorder characterized by boné marrow failure, developmental malformations and congenital abnormalities. FA progressive bone marrow failure usually evolves to aplastic anemia and malignant neoplasias, especially acute myeloid leukemia and head and neck squamous cell carcinomas. The risk of malignancies is increased after bone marrow transplantation (BMT), and recently the role of human papillomavirus (HPV) in FA carcinogenesis was proposed. We performed an oral examination, liquid based cytology sampling of oral epithelial cells, assessment of atypias according to Papanicolaou classification, cytomorphometric analysis of oral epithelial cells, HPV screening by PCR and genotyping by reverse hybridization. Sample subjects were divided into 4 groups: BMT FA patients (I), not BMT FA patients (II), BMT severe aplastic anemia patients (SAA) and a healthy control group (IV). No malignant lesions were detected on oral examination. In comparison with the control group the results showed: a) an increase in the nuclear area (NA) in oral epithelial cells in FA and SAA (p<0.05); b) an increase in the cytoplasmic area (CA) in FA (p<0.05); c) a decrease in the NA/CA ratio in BMT FA patients (p<0.05), an increase of it in SAA (p<0.05) and no alterations in not BMT FA patients; d) a high HPV prevalence was detected in BMT FA (35%, p<0.05) and SAA patients (38%, p<0.05), whereas in not BMT FA patients (27.27%) there was not a significant statistical difference in comparison with the control group (6.25%) and e) the high risk, HPV-16, was the most frequent genotype (group I 71.42%, group II 33.33%, group III 28.57%, group IV 0%). HPV infection odds ratio adjusted for age and years of BMT showed an increase in HPV infection for both factors. In conclusion, results demonstrated that there were morphometric alterations in oral epithelial cells in FA and a high prevalence of oral HPV, especially HPV-16, in these patients without malignant lesions.