Orientador: Prof. Dr. Cassius Carvalho Torres-Pereira / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Odontologia. Defesa: Curitiba, 2016 / Inclui referências : f. 69-79 / Área de concentração / Resumo: A anemia de Fanconi (AF) é uma síndrome genética rara caracterizada por instabilidade cromossômica e dificuldade de reparo do DNA. Pacientes com AF desenvolvem o carcinoma de células escamosas (CCE) na boca mais cedo e com maior frequência que a população em geral, especialmente após o transplante de células tronco hematopoéticas (TCTH). Embora tenha aumentado a evidência de um papel etiológico da microbiota local e o processo de carcinogênese; não existem informações sobre a microbiota bucal de pacientes com AF. O objetivo desse estudo foi explorar a microbiota salivar de 61 pacientes com AF e comparar os resultados com a condição de saúde bucal e fatores de riscos associados ao desenvolvimento do CCE. Depois de responder a um questionário e ser submetido a um exame clínico intrabucal, todos os pacientes passaram por um etapa de coleta de saliva e as amostras foram analisadas utilizando o sequenciamento do gene 16S rRNA (Região Hipervariável :V3-V4 Miseq, Illumina). O perfil microbiano associado aos parâmetros clínicos e dados retirados de prontuário médico foram analisados utilizando modelos lineares. A mediana de idade da amostra estudada foi de 22 anos e a maioria deles haviam sido submetidos ao TCTH (n=53). Os filos bacterianos mais abundantes foram Firmicutes (média da abundância relativa ± desvio padrão(DP)) (42,1% ± 10,1%) e Bacteroidetes (25,4% ± 11,4%). O histórico de doença do enxerto contra o hospedeiro (DECH) bucal (n=27) estava associada com maiores proporções de Firmicutes (43,8% x 38,5%, p = 0,05) quando comparados com os que não apresentaram DECH. Altos níveis de sangramento gengival foram associados com os genêros Prevotella (22,25% x 20%), Streptococcus (19,83% x 17,61%), Porphyromonas (3,63% x 1,42%, p = 0,03), Treponema (1,02% x 0,28%, p = 0,009), Parvimonas (0,28% x 0,07%, p = 0,02) e Dialister (0,27% x 0,10%, p = 0,04). Por fim, pacientes transplantados à mais de 11 anos mostraram níveis mais elevados de Streptococcus (18,4%), Haemophilus (12,7%) e Neisseria (6,8%). Em conclusão, pacientes com AF que apresentavam piores condições de higiene bucal abrigavam maiores proporções de gêneros de bactérias compatíveis com doença periodontal. Foram observadas diferenças microbianas específicas, na presença de longo tempo de transplante, histórico de GVHD e mucosite, bem como, na presença de lesões com potencial de malignidade. Palavras-chave: Anemia de Fanconi. Câncer bucal. Microbiota. Saliva. Bactéria. Sequenciamento de Nucleotídeos de Alto Rendimento. / Abstract: Fanconi anemia (FA) is a rare genetic disease characterized by chromosomal instability and impaired DNA damage repair. FA patients develop oral squamous cell carcinoma (OSCC) earlier and more frequently than the general population, especially after hematopoeitic stem cell transplantation (HSCT). Although evidence of an etiological role of the local microbiome and carcinogenesis has grown, no information exists regarding the oral microbiome of FA patients. The aim of this study was to explore the salivary microbiome of 61 FA patients regarding their oral health status and OSCC risk factors. After answering a questionnaire and receiving oral clinical examination, saliva samples were collected and analyzed using 16rRNA sequencing (V3-V4 hypervariable region, MiSeq, Illumina). The microbial profiles associated with medical and clinical parameters were analyzed using general linear models. Patients were young (mean age = 22 yrs old) and most of them had received HSCT (n=53). The most abundant phyla were Firmicutes (mean relative abundance ± SD) (42.1%±10.1%) and Bacteroidetes (25.4%±11.4%). A history of graft-versus-host disease (GVHD) (n=27) was associated with higher proportions of Firmicutes (43.8% x 38.5%, p=0.05). High levels of gingival bleeding were associated with the genera Prevotella (22.25% x 20%), Streptococcus (19.83% x 17.61%), Porphyromonas (3.63% x 1.42%, p=0.03), Treponema (1.02% x 0.28%, p=0.009), Parvimonas (0.28% x 0.07%, p=0.02) and Dialister (0.27% x 0.10%, p=0.04). Finally, participants transplanted longer than 11 years showed highest levels of Streptococcus (18.4%), Haemophilus (12.7%) and Neisseria (6.8%). In conclusion, FA patients with poor oral hygiene harbored higher proportions of genera of bacteria compatible with gingival disease. Specific microbial differences were observed in the presence of a history of long time since HSCT, history of oral GVHD and mucositis as well when potential malignant oral lesion was present. Keywords: Fanconi anemia. Oral cancer. Microbiome. Saliva. Bacteria. Massively-Parallel Sequencing.

Resumo: A Anemia de Fanconi (AF) é uma doença genética rara caracterizada por instabilidades cromossômicas que geram anormalidades somáticas de graus variáveis, falência medular progressiva e susceptibilidade aumentada a neoplasias. O único tratamento curativo é o transplante de células tronco hematopoéticas (TCTH), entretanto a toxicidade e falha de pega ainda são limitantes para sua realização. A falha de pega do enxerto ou rejeição é uma complicação grave e potencialmente fatal que ocorre em até 30% dos pacientes com AF submetidos ao TCTH, dependendo do tipo de doador e regime de condicionamento utilizado. Entre Janeiro de 1985 e Outubro de 2011, 238 pacientes com AF realizaram TCTH nessa instituição. Duzentos e doze pacientes foram avaliáveis quanto à pega medular e divididos em 3 grupos. O grupo 1 foi constituído por 25 pacientes que tiveram falha primária de pega (FPP). O grupo 2 por 9 pacientes com falha secundária de pega (FSP) ou evolução para leucemia e o grupo 3, com 178 casos com pega medular adequada. Os pacientes com falha primária e secundária de pega apresentaram maior duração de doença e maior número de transfusões sanguíneas prévias ao TCTH do que o grupo com pega medular (p=0,001 e p<0,001). Doador não aparentado (NAP) foi utilizado em 84% dos pacientes do G1 e apenas 33% do G3 (p<0,001); houve ainda menor número de transplantes totalmente compatíveis no G1 do que nos outros grupos (p<0,001). A fonte de células foi sangue de cordão umbilical (SCU) em 56% dos pacientes do G1 e 13% no G3. Na análise multivariada os fatores associadas a uma maior FPP foram maior número de transfusões (p=0,003), a utilização de doadores NAP (p=0,001) ou com incompatibilidades HLA (p=0,025) ou a ausência de fludarabina no regime de condicionamento (p=0,005). O fator predisponente para pega medular não sustentada ou evolução para leucemia foi maior número de transfusões (p=0,032). Vinte e quatro pacientes foram submetidos a um novo TCTH, sendo 20 do G1 e 4 do G2 e apenas seis pacientes estão vivos. A incidência cumulativa para a falha de pega foi de 18,9%, sendo maior nos transplantes com doadores NAP (36,2%) e com SCU (40%). A falha de pega ou rejeição é uma complicação extremamente grave e mais frequente nos transplantes não aparentados, com incompatibilidades e em pacientes mais transfundidos. Novos regimes de condicionamento, imunomodulação ou redução do tempo para o encontro de doadores NAP podem ser alternativas na tentativa de reduzir a incidência da rejeição nos pacientes com AF.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22002号 / 医博第4516号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 松田 文彦, 教授 山田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18906号 / 医科博第62号 / 新制||医科||4(附属図書館) / 31857 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 山下 潤, 教授 野田 亮, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The dawn of the genetic era has allowed for investigation of gene transfer therapy as a treatment for certain diseases. Fanconi anemia (FA) is a rare genetic disorder in which the majority of patients develops progressive bone marrow failure (BMF) and require bone marrow transplantation. A possible alternative treatment is autologous gene therapy; however, original clinical trials involving gene transfer for FA were unsuccessful. This has led to re-evaluation of the gene transfer protocols, the vectors and also a deeper investigation of the FA pathway itself. My work has focused on illuminating these areas to further advance gene transfer therapy for FA. Many gene transfer protocols require the hematopoietic stem and progenitor cells (HSC/HPC) to be collected and then transduced ex vivo. The most common collection method is mobilization of the HSC/HPC to the peripheral blood (PB) using granulocyte colony-stimulating factor (G-CSF) and collection via apheresis. In FA patients G-CSF fails to mobilize a sufficient number of HSC/HPC. This has led to research into agents such as AMD3100, a CXCR4 antagonist, which may replace or augment G-CSF mobilization. These data show in two FA murine models that AMD3100 synergizes with G-CSF resulting in a significant increase in mobilization as compared to G-CSF alone. Previous work in our lab has shown that prototype foamy virus (FV) is an efficient gene transfer vector. Here a modified FV vector is used to transduce mobilized FA cells. The data indicate that long-term repopulating cells mobilized with both G-CSF and AMD3100 can be efficiently transduced by our FV vector. Clinically, FA is characterized mainly by BMF, but also by myelodysplasia (MDS) and acute myeloid leukemia (AML). However, current FA murine models do not display these disease phenotypes. These data show that double-mutant Fancc-/-;Fancg-/- mice spontaneously develop BMF, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous BMF and MDS in FA and novel gene transfer protocols for FA.

Since the dawn of the genomics era, the genetics of numerous human disorders has been understood which has led to improvements in targeted therapeutics. However, the focus of most research has been primarily on protein coding genes, which account for only 2% of the entire genome, leaving much of the remaining genome relatively unstudied. In particular, repetitive sequences, called microsatellites (MST), which are tandem repeats of 1 to 6 bases, are known to be mutational hotspots and have been linked to diseases, such as Huntington disease and Fragile X syndrome. This work represents a significant effort towards closing this knowledge gap. Specifically, we developed a next generation sequencing based enrichment method along with the supporting computational pipeline for detecting novel MST sequences in the human genome. Using this global MST enrichment protocol, we have identified 790 novel sequences. Analysis of these novel sequences has identified previously unknown functional elements, demonstrating its potential for aiding in the completion of the euchromatic DNA. We also developed a disease risk diagnostic using a novel target specific enrichment method that produces high resolution MST sequencing data that has the potential to validate, for the first time, the link between MST genotype variation and cancer. Combined with publicly available exome datasets of non-small cell lung cancer and 1000 genomes project, the target specific MST enrichment method uncovered a signature set of 21 MST loci that can differentiate between lung cancer and non-cancer control samples with a sensitivity ratio of 0.93. Finally, to understand the molecular causes of MST instability, we analyzed genomic variants and gene expression data for an autosomal recessive disorder, Fanconi anemia (FA). This first of its kind study quantified the heterogeneity of FA cells and demonstrated the possibility of utilizing the DNA crosslink repair dysfunctional FA cells as a suitable system to further study the causes of MST instability. / Ph. D. / The field of genetics has enjoyed substantial growth since the conclusion of the human genome project, which was declared complete in the year 2003. The human genome project produced the first framework for the human DNA sequence, the human genome. With the availability of this framework, the understanding of the genetic basis for a number of diseases has significantly grown, which has resulted in better methods of clinical diagnosis and treatment. While the current focus on understanding the genomic regions that are responsible for making proteins has inarguably helped, it has also created a gap in knowledge. Protein coding regions of the human genome account only for 2% of the entire human genome and a large part (47%) of the genome is occupied by repetitive DNA. DNA sequences can be complex, with the nucleotides arranged in no particular order, e.g. ATCGTACGA, or DNA sequences can be repetitive, e.g. ATATATATAT. Repetitive sequences, which have repeating units of 1 to 6 bases, are called microsatellites (MST). MSTs have been shown to be unstable and they have been linked to diseases such as Huntington disease and Fragile X syndrome. This work helps to close this knowledge gap by developing molecular methods and computational tools focused on identifying MST variations. Research conducted with this aim has resulted in three major accomplishments. One, we developed novel molecular and computational methods which we used to detect 790 previously unknown sequences in the human genome. This work proved the ability of our method to uncover functional elements in the human genome that can potentially answer numerous biological questions. Two, we developed another novel method for the production of high resolution MST sequence data that not only can improve MST research in general but also shows the potential for the development of new genetic diagnostics and cancer therapeutics. We identified a signature set of 21 MST sequences that can differentiate between lung cancer patient genomes and non-cancer control genomes. These results represent the first potential validation for a proposed link between MST sequence length (genotype) variation and cancer. Three, we attempt to understand a possible molecular cause and consequences of MST instability in a disease called Fanconi anemia. The results from this work not only, for the first time, quantify the effects of this disease on the genome but also establishes Fanconi anemia as a suitable system for studying MST instability in detail.

L'anémie de Fanconi (AF) est une maladie génétique rare. Chez l’homme, toute mutation dans l’une des 22 protéines du complexe de Fanconi entraîne une insuffisance médullaire et une prédisposition au cancer. Cette pathologie est également caractérisée par divers défauts de développement, dont une petite taille et des malformations squelettiques des membres supérieurs et inférieurs. En effet, plus de la moitié des enfants atteints de l’AF ont des anomalies radiales (radial-ray anomalies) couplées à une tendance précoce à l’ostéoporose. Cependant, les mécanismes sous-jacents conduisant à des défauts osseux dans l’AF restent inexpliqués. Notre laboratoire a mis en évidence la surexpression de Dickkopf-1 (DKK1), un inhibiteur de la voie de Wnt, dans le plasma des souris Fanconi. Cette protéine est impliquée dans le développement des membres et l’activité ostéoblastique. De plus, sa surexpression dans le plasma corrèle avec une diminution de la densité minérale osseuse chez les humains. La surexpression de DKK1 pourrait donc refléter une altération du système squelettique chez les souris Fanconi. Ce manuscrit présente le travail que j’ai réalisé dans le laboratoire de Madeleine Carreau au cours de mon doctorat afin de caractériser le développement squelettique embryonnaire des souris Fanconi et déterminer les mécanismes responsables de l'altération du développement et du métabolisme osseux chez les souris adultes. À ces fins, une double coloration à l’alizarine et au bleu Alcian a été réalisée sur des embryons de souris FancC - / -, FancA - / - et de souris de type sauvage E15.5 à 19.5 day post conception (dpc) pour évaluer leur maturation squelettique. Chez les adultes, la structure et le contenu minéral osseux ont été évalués à l’aide d’analyses de micro-computed tomography (μ- CT) sur des tibias provenant de souris FancC - / - et de souris sauvages. Des analyses histomorphométriques ont été effectuées pour évaluer la capacité de formation osseuse des ostéoblastes et la résistance osseuse a été évaluée en utilisant un test de flexion en trois points. De plus, des cultures in vitro ont été réalisées pour évaluer la capacité de différenciation des cellules souches mésenchymateuses et des analyses des transcrits sur des cellules osseuses et de la moelle osseuse ont été réalisées pour identifier les mécanismes moléculaires conduisant à une altération de la physiologie osseuse. Nos résultats montrent que les embryons des souris FancC - / - et FancA - / - présentent une forte diminution de la minéralisation de leur squelette indiquant un développement squelettique anormal chez ces souris. Les souris FancC -/- adultes présentent une diminution de la densité minérale osseuse associée à une diminution de la résistance osseuse chez les mâles. Grâce aux études in vitro, nous avons pu constater que les cellules souches mésenchymateuses de la moelle osseuse FancC - / - et FancA - / - ont une capacité de différenciation ostéoblastique altérée et présentent un biais de différenciation en faveur de l’adipogenèse. Ces résultats sont associés à l’altération des profils d’expression génique des cellules osseuses. Nos résultats suggèrent que la physiologie osseuse défectueuse dans l’AF survient in utero et résulte éventuellement d’une altération de la fonction des cellules souches mésenchymateuses de la moelle osseuse. Ces résultats fournissent, pour la première fois, des informations précieuses sur les mécanismes impliqués dans les défauts de développement dans l’AF. Nos résultats renforcent le lien important entre le comportement des cellules souches hématopoïétiques et l’altération du métabolisme osseux dans cette maladie. De futures études devraient se concentrer sur ce domaine pour mieux comprendre les mécanismes des défauts osseux et espérer un traitement limitant l’altération du tissu osseux dans l’AF. / Fanconi Anemia (FA) is a rare genetic disease. In humans, any mutation in one of the 22 proteins of the Fanconi complex leads to bone marrow failure and cancer predisposition. This pathology is also characterized by various developmental defects including short stature and skeletal malformations of the upper and lower limbs. Indeed, more than half of children affected with FA have radial-ray abnormalities with a tendency to develop early osteoporosis. However, the underlying mechanisms leading to bone defects in FA remains elusive. Previous results from our laboratory showed that Fanconi mice overexpress the Wnt signaling pathway inhibitor Dickkopf-1 (DKK1) in their plasma. This protein is implicated in limb development and osteoblast activity and its overexpression in plasma correlates with a decrease in bone mineral density in humans. Therefore, DKK1 overexpression could reflect an alteration of the skeletal system in Fanconi mice. This manuscript presents the work I achieved in Madeleine Carreau’s lab to characterize the embryonic skeletal development of Fanconi mice and determine the mechanisms leading to altered bone development and metabolism in adult mice. To this aim, alizarin red and Alcian blue double staining was performed on mouse embryos (E15.5 to 19.5 dpc) to evaluate skeletal maturation. In adults, bone structure and mineral content were evaluated using μCT-scan analyses of tibias from FancC-/- and wild-type mice. Histomorphometric analyses were performed to assess bone forming abilities of osteoblasts and bone stiffness was evaluated using three points bending test. In vitro cultures were performed to assess mesenchymal stem cell differentiation ability and q-PCR analysis of bone and marrow cells were performed to identify molecular mechanisms leading to altered bone physiology. Our results show that FancC-/- and FancA-/- embryos have an abnormal skeletal development indicated by a twenty percent decrease of bone mineralization surface. In adults, FancC-/- mice present a decrease in bone mineral density associated with a decrease in male’s bone stiffness. Using in vitro studies, we found that FancC-/- and FancA-/- bone marrow-derived mesenchymal stem cells (BM-MSCs) have reduced osteoblastic differentiation capabilities and favor adipogenesis. Those results were associated with the alteration of bone cells gene expression profiles. Our results suggest that defective bone physiology in FA occurs in utero and possibly results from altered BM-MSCs function. These results provide, for the first time, valuable insights into the mechanisms involved in FA developmental defects. Our results strengthen the important link between hematopoietic stem cells behavior and bone metabolism alteration in this disease. Future studies should focus on this area to better understand the mechanisms of bone defects and hope for targeted treatment for Fanconi Anemia.

L'anémie de Fanconi est une maladie génétique récessive rare, caractérisée par une défaillance de la moelle osseuse, des anomalies du développement et une forte incidence de cancer, essentiellement des leucémies myéloïdes aiguës. L'anémie de Fanconi peut être causée par la mutation de 13 gènes différents, et neuf des protéines Fane sont responsables de la monoubiquitination de FANCD2, qui est donc centrale dans la 'voie Fanconi'. Le complexe MRN joue plusieurs rôles essentiels dans la réparation des cassures double-brin dans l'ADN par recombinaison homologue : dans la signalisation du dommage et dans les points de contrôle du cycle cellulaire, dans la préparation des extrémités de la cassure pour les étapes suivantes de la réparation ainsi qu'un rôle structural afin de faciliter la réparation. Il a été montré précédemment que la protéine FANCD2 colocalise avec NBS1. Cependant, la relation fonctionnelle entre FANCD2 et MRE11 est mal comprise. Mes travaux de doctorat montrent que l'inhibition de MRE11, NBS1 ou RAD50 conduit à la déstabilisation de FANCD2. FANCD2 s'accumule de la phase S à la phase G2 aux sites contenant de l'ADN simple brin ou aux sites de dommages à l'ADN. La protéine FANCD2 purifiée lie préférentiellement d'ADN simple brin en comparaison à différents substrats d'ADN. L'inhibition de l'activité nuclease de MRE11 par le MIRIN diminue le nombre de foyers de FANCD2 formés in vivo. Nous proposons donc que FANCD2 lie l'ADN simple brin provenant de la résection de la cassure double brin par MRE11. Nos données établissent que MRN est un régulateur essentiel de la stabilité et de la fonction de FANCD2 au cours de la réponse aux dommages à l'ADN.