Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Introdução: A síndrome de Bloom (SB) é uma síndrome de instabilidade cromossômica rara, transmitida por herança autossômica recessiva. Caracteriza-se por deficiência de crescimento pré e pós-natal, microcefalia, hipoplasia malar, eritema telangiectásico em face e comprometimento do sistema imunológico. Os pacientes com SB apresentam predisposição aumentada para o desenvolvimento de neoplasias em idade precoce, sendo esta, a principal causa de óbito. No estudo citogenético observa-se aumento de quebras cromossômicas espontâneas e trocas entre cromátides irmãs (TCI), que é utilizada como marcador diagnóstico para a SB. Essas alterações são causadas por um defeito no mecanismo de reparo do DNA, decorrente de uma mutação no gene BLM. Objetivos: Realizar o estudo citogenético de trocas entre cromátides irmãs para o diagnóstico de pacientes com suspeita clínica de SB; caracterizar os aspectos clínicos e avaliar a evolução de pacientes com SB. Métodos: Foram estudados nove pacientes (4 M e 5 F) pertencentes a oito famílias com suspeita clínica de SB utilizando preparações cromossômicas tratadas com 5- bromo-2-desoxiuridina (BrdU) e coloração Hoechst - Giemsa para visualização diferencial das cromátides irmãs e análise de freqüência de TCI. Resultados e Discussão: Todos os pacientes foram positivos para a pesquisa de TCI cuja freqüência variou de 45,2 a 61,3 TCI/metáfase. A idade dos pacientes ao diagnóstico variou de 1a1m até 11a (média de 4a6m). O principal motivo do encaminhamento foi o déficit de crescimento e apenas um paciente foi encaminhado por apresentar lesões cutâneas. Todos apresentaram deficiência de crescimento pré e pós-natal, microcefalia e hipoplasia malar. O eritema esteve presente em 8/9 pacientes. Manchas café-au-lait e/ou manchas hipocrômicas foram observadas em sete pacientes. Um paciente apresentou agenesia unilateral da fíbula, encurtamento da tíbia e agenesia do 5° artelho, associado à hipoplasia renal. As infecções de repetição foram relatadas em 8/9 pacientes, sendo principalmente pneumonia e diarreia. Deficiência de imunoglobulinas foi observada em 6/9 pacientes, principalmente: deficiência de IgG (3/6), de IgA (2/6) e de IgM (1/6). A consanguinidade entre os pais foi encontrada em 4/8 famílias, apenas uma família apresentou dois filhos afetados. Duas pacientes (2/9) evoluíram com tumor de Wilms (TW), uma aos 3a6m e a outra aos 3a11m. Houve recidiva em uma paciente que faleceu aos cinco anos. A outra paciente evoluiu bem e atualmente está com 20 anos. Conclusão: O diagnóstico da SB deve ser feito precocemente baseado na avaliação clínica. A pesquisa citogenética de TCI, que é de baixo custo e fácil aplicação, é fundamental para a confirmação diagnóstica. A freqüência aumentada para o desenvolvimento de neoplasias em idade precoce, alerta para um rastreamento das neoplasias mais comuns como linfoma, leucemia e tumor de Wilms. / Introduction: Bloom syndrome (BS) is a rare chromosomal instability syndrome, transmitted by autosomal recessive inheritance. It´s characterized by pre and postnatal growth deficiency, microcephaly, malar hypoplasia, telangiectatic erythema on the face and impaired immune system. BS patients present an increased predisposition to develop cancer at early age, which is the main cause of death. In the cytogenetic exam is observed an increase of spontaneous breaks and sister chromatid exchange (SCE) that is used as a diagnostic biomarker for the BS. These changes are caused by a defect in DNA repair mechanism, due to mutations in the BLM gene. Objectives: Perform the cytogenetic study of sister chromatid exchange for the clinical diagnosis of BS patients; to characterize the clinical aspects and assess the follow-up of patients. Methods: Nine patients (4 M, 5 F) from eight families with clinical diagnoses of BS were studied using standard chromosome preparations treated with 5-bromo-2-deoxyuridine (BrdU) and Hoechst-Giemsa differential staining for visualization and analysis of frequency of SCE. Results and Discussion: All patients were positive for the presence of SCE with the frequency ranged from 45.2 to 61.3 SCE/metaphase. The age at diagnosis ranged from 1y1mo to 11y (mean 4y6mo). The main reason for referral was growth deficit except one due to skin lesions. All patients presented pre and post-natal growth deficiency, microcephaly and malar hypoplasia. The erythema was present in 8/9 patients. Cafe-au-lait spots and/or hypochromic spots were observed in seven patients. One patient had unilateral agenesis of the fibula, shortening tibia and agenesis of the 5th toe associated with renal hypoplasia. The recurrent infections were reported in 8/9 patients, mainly pneumonia and diarrhea. Immunoglobulin deficiency was observed in 6/9 patients such as IgG (3/6), IgA (2/6) and IgM (1/6). The parental consanguinity was found in 4/8 families, one family had two affected. Two patients (2/9) developed Wilms tumor (WT), one at 3y6mo and another at 3y11mo. There was recurrence in one patient who died at five years. The other patient is well at 20 years old. Conclusion: The diagnosis of BS should be done early based in clinical findings. The cytogenetic for SCE exam is essential for diagnostic confirmation, which is low cost and easy application. The screening for the most common malignancies such as lymphoma, leukemia and WT must be done due to increased predisposition for cancer development at an early age

Introdução: A síndrome de Bloom (SB) é uma síndrome de instabilidade cromossômica rara, transmitida por herança autossômica recessiva. Caracteriza-se por deficiência de crescimento pré e pós-natal, microcefalia, hipoplasia malar, eritema telangiectásico em face e comprometimento do sistema imunológico. Os pacientes com SB apresentam predisposição aumentada para o desenvolvimento de neoplasias em idade precoce, sendo esta, a principal causa de óbito. No estudo citogenético observa-se aumento de quebras cromossômicas espontâneas e trocas entre cromátides irmãs (TCI), que é utilizada como marcador diagnóstico para a SB. Essas alterações são causadas por um defeito no mecanismo de reparo do DNA, decorrente de uma mutação no gene BLM. Objetivos: Realizar o estudo citogenético de trocas entre cromátides irmãs para o diagnóstico de pacientes com suspeita clínica de SB; caracterizar os aspectos clínicos e avaliar a evolução de pacientes com SB. Métodos: Foram estudados nove pacientes (4 M e 5 F) pertencentes a oito famílias com suspeita clínica de SB utilizando preparações cromossômicas tratadas com 5- bromo-2-desoxiuridina (BrdU) e coloração Hoechst - Giemsa para visualização diferencial das cromátides irmãs e análise de freqüência de TCI. Resultados e Discussão: Todos os pacientes foram positivos para a pesquisa de TCI cuja freqüência variou de 45,2 a 61,3 TCI/metáfase. A idade dos pacientes ao diagnóstico variou de 1a1m até 11a (média de 4a6m). O principal motivo do encaminhamento foi o déficit de crescimento e apenas um paciente foi encaminhado por apresentar lesões cutâneas. Todos apresentaram deficiência de crescimento pré e pós-natal, microcefalia e hipoplasia malar. O eritema esteve presente em 8/9 pacientes. Manchas café-au-lait e/ou manchas hipocrômicas foram observadas em sete pacientes. Um paciente apresentou agenesia unilateral da fíbula, encurtamento da tíbia e agenesia do 5° artelho, associado à hipoplasia renal. As infecções de repetição foram relatadas em 8/9 pacientes, sendo principalmente pneumonia e diarreia. Deficiência de imunoglobulinas foi observada em 6/9 pacientes, principalmente: deficiência de IgG (3/6), de IgA (2/6) e de IgM (1/6). A consanguinidade entre os pais foi encontrada em 4/8 famílias, apenas uma família apresentou dois filhos afetados. Duas pacientes (2/9) evoluíram com tumor de Wilms (TW), uma aos 3a6m e a outra aos 3a11m. Houve recidiva em uma paciente que faleceu aos cinco anos. A outra paciente evoluiu bem e atualmente está com 20 anos. Conclusão: O diagnóstico da SB deve ser feito precocemente baseado na avaliação clínica. A pesquisa citogenética de TCI, que é de baixo custo e fácil aplicação, é fundamental para a confirmação diagnóstica. A freqüência aumentada para o desenvolvimento de neoplasias em idade precoce, alerta para um rastreamento das neoplasias mais comuns como linfoma, leucemia e tumor de Wilms. / Introduction: Bloom syndrome (BS) is a rare chromosomal instability syndrome, transmitted by autosomal recessive inheritance. It´s characterized by pre and postnatal growth deficiency, microcephaly, malar hypoplasia, telangiectatic erythema on the face and impaired immune system. BS patients present an increased predisposition to develop cancer at early age, which is the main cause of death. In the cytogenetic exam is observed an increase of spontaneous breaks and sister chromatid exchange (SCE) that is used as a diagnostic biomarker for the BS. These changes are caused by a defect in DNA repair mechanism, due to mutations in the BLM gene. Objectives: Perform the cytogenetic study of sister chromatid exchange for the clinical diagnosis of BS patients; to characterize the clinical aspects and assess the follow-up of patients. Methods: Nine patients (4 M, 5 F) from eight families with clinical diagnoses of BS were studied using standard chromosome preparations treated with 5-bromo-2-deoxyuridine (BrdU) and Hoechst-Giemsa differential staining for visualization and analysis of frequency of SCE. Results and Discussion: All patients were positive for the presence of SCE with the frequency ranged from 45.2 to 61.3 SCE/metaphase. The age at diagnosis ranged from 1y1mo to 11y (mean 4y6mo). The main reason for referral was growth deficit except one due to skin lesions. All patients presented pre and post-natal growth deficiency, microcephaly and malar hypoplasia. The erythema was present in 8/9 patients. Cafe-au-lait spots and/or hypochromic spots were observed in seven patients. One patient had unilateral agenesis of the fibula, shortening tibia and agenesis of the 5th toe associated with renal hypoplasia. The recurrent infections were reported in 8/9 patients, mainly pneumonia and diarrhea. Immunoglobulin deficiency was observed in 6/9 patients such as IgG (3/6), IgA (2/6) and IgM (1/6). The parental consanguinity was found in 4/8 families, one family had two affected. Two patients (2/9) developed Wilms tumor (WT), one at 3y6mo and another at 3y11mo. There was recurrence in one patient who died at five years. The other patient is well at 20 years old. Conclusion: The diagnosis of BS should be done early based in clinical findings. The cytogenetic for SCE exam is essential for diagnostic confirmation, which is low cost and easy application. The screening for the most common malignancies such as lymphoma, leukemia and WT must be done due to increased predisposition for cancer development at an early age

La stabilité du génome est compromise par les déséquilibres du pool de dNTPs qui affectent la vitesse de progression des fourches de réplication. Par exemple, la déficience en cytidine désaminase (CDA) conduit à un excès de dCTP qui induit un ralentissement des fourches de réplication. Les résultats obtenus au cours de ma thèse ont permis de mettre en évidence un nouveau mécanisme par lequel un déséquilibre du pool de nucléotides compromet la complétion de la réplication et la ségrégation correcte des chromosomes. En utilisant des techniques de peignage moléculaire, de microscopie électronique et d’imagerie cellulaire permettant de quantifier le niveau basal de PAR, nous avons montré que la réplication incomplète de l’ADN lorsque la CDA est absente est due à l’inhibition partielle de PARP-1, et n’est pas liée au ralentissement de la vitesse de progression des fourches de réplication. En effet, l’accumulation intracellulaire de dCTP inhibe l’activité de PARP-1 ce qui réduit l’activation de Chk1 et l’efficacité des points de contrôle situés en aval, favorisant ainsi l’accumulation de séquences d’ADN non répliquées en mitose. Celles-ci conduisent alors à la formation de ponts anaphasiques ultrafins (UFBs) entre les chromatides sœurs au niveau de sites difficiles à répliquer tels que les centromères et les sites fragiles. Ces résultats ont des implications directes dans le syndrome de Bloom (BS), une maladie génétique rare combinant prédisposition aux cancers et instabilité génétique. Ce syndrome est la conséquence de la mutation du gène BLM, codant pour une hélicase RecQ du même nom. La déficience en BLM conduit à une chute de l’expression de la CDA résultant en une augmentation des UFBs entièrement due à l’inhibition de PARP-1 par la dCTP, indépendamment de BLM. Ces travaux décrivent ainsi une conséquence pathologique encore inconnue du déséquilibre du pool de nucléotides et révèlent un rôle inattendu de PARP-1 dans la surveillance des séquences d’ADN non répliquées prévenant leur accumulation en mitose et les défauts de ségrégation des chromosomes associés. / Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that in CDA-deficient cells DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. Indeed, the intracellular accumulation of dCTP inhibits PARP-1 activity compromising the activation of Chk1 and the downstream checkpoints efficiency, allowing the subsequent accumulation of unreplicated DNA in mitosis. This unreplicated DNA leads to the formation of ultrafine anaphase bridges (UFBs) between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of UFBs due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our results describe previously unknown pathological consequences of the distortion of dNTP pools and reveal an unexpected role for PARP-1 in preventing unreplicated DNA accumulation in mitosis and in preventing chromosome segregation defects.

Les cassures double brin de l’ADN (CDB) sont des lésions qui peuvent conduire à des réarrangements génétiques. Deux voies sont impliquées dans la réparation de ces dommages: la recombinaison homologue (HR) et la ligature d’extrémités nonhomologues (NHEJ).Au laboratoire un substrat intrachromosomique permettant de mesurer l’efficacité et la fidélité du NHEJ à été mis en place (Guirouilh-Barbat 2004). Cette approche a permis de démontrer l’existence d’une voie alternative à KU qui utilise des microhomologies présentes de part et d’autre de la cassure - le NHEJ alternatif (Guirouilh-Barbat 2004, Guirouilh-Barbat et Rass 2007). Les travaux de ma thèse consistent à caractériser les principaux acteurs de cette voie. En absence de KU, cette voie alternative du NHEJ, s'initierait tout d’abord parla résection d'extrémités d’ADN non protégées. Nous avons montré que l’activité nucléasique de MRE11 est nécessaire à ce mécanisme. La surexpression de MRE11 conduit à une stimulation du NHEJ, contrairement à l’extinction de la protéine par siRNA, résultant en une baisse de son efficacité de deux fois. Nos résultats montrent également que les protéines RAD50 et CtIP agissent dans la même voie que MRE11. De plus, dans les cellules déficientes pour XRCC4, la MIRIN – un inhibiteur du complexe MRN - conduit à une chute de l'efficacité de la réparation, démontrant le rôle de MRE11 dans la voie alternative du NHEJ. Nous avons aussi montré que MRE11 peut agir de manière dépendante et indépendante de la kinase ATM (Rass et Grabarz, Nat Struct Mol Biol 2009). L'initiation de la résection de la cassure doit être ensuite poursuivie par une dégradation plus importante de l'ADN qui est assuré par les protéines Exo1 et Sgs1/Dna2 chez la levure. Chez les mammifères, des études in vitro suggèrent un modèle similaire à deux étapes. Nous avons choisi de nous intéresser au rôle de la protéine BLM, qui est l’un des homologues humains de la RecQ hélicase Sgs1, dans la résection. Nos expériences montrent que l’absence de BLM diminue l’efficacité du NHEJ. De plus, l’extinction de BLM conduit à une augmentation d’évènements infidèles lors de la réparation par NHEJ et l’apparition d’évènements de résection de grande taille (>200nt). Ceci suggère que BLM protège contre de longues résections lors de la mise en place du NHEJ alternatif. De manière cohérente, BLM est impliquée dans la protection contre la résection dépendante de CtIP lors des étapes précoces de la recombinaison homologue. En conclusion, nos résultats montrent un rôle prédominant de BLM dans la protection contre un excès de résection médiée par CtIP. BLM interagit avec 53BP1 aux sites de dommages de manière dépendante d’ATM afin de réguler le processus de résection, en contrecarrant l’action de BRCA1. Ceci souligne à nouveau le rôle essentiel de BLM dans la protection contre la résection et la favorisation de la conversion génique sans crossing-over, ce qui est primordial pour le maintien de la stabilité du génome. / DNA double strand breaks (DSBs) are highly cytotoxic lesions, which can lead to genetic rearrangements. Two pathways are responsible for repairing these lesions : homologous recombination (HR) and non homologous end joining (NHEJ). In our laboratory, an intrachromosomal substrate has been established in order to measure the efficiency and the fidelity of NHEJ in living cells (Guirouilh-Barbat 2004). This approach led us to identify a KU-independent alternative pathway, which uses microhomologies in the proximity of the junction to accomplish repair – the alternative NHEJ (Guirouilh-Barbat 2004, Guirouilh-Barbat et Rass 2007). The goal of my thesis consisted in identifying and characterising major actors of this pathway. In the absence of KU, alternative NHEJ would be initiated by ssDNA resection of damaged ends. We showed that the nuclease activity of MRE11 is necessary for this mechanism. MRE11 overexpression leads to a two fold stimulation of NHEJ efficiency, while the extinction of MRE11 by siRNA results in a two fold decrease. Our results demonstrate that the proteins RAD50 and CtIP act in the same pathway as MRE11. Moreover, in cells deficient for XRCC4, MIRIN – an inhibitor of the MRN complex – leads to a decrease in repair efficiency, implicating MRE11 in alternative NHEJ. We also showed that MRE11 can act in an ATM-dependent and independent manner (Rass et Grabarz Nat Struct Mol Biol 2009). The initiation of break resection needs to be pursued by a more extensive degradation of DNA, which is accomplished in yeast by the proteins Exo1 and Sgs1/Dna2. In human cells, in vitro studies have recently proposed a similar model of a two-step break resection. We chose to elucidate the role of one of the human homologs of Sgs1 – the RecQ helicase BLM – in the resection process. Our experiments show, that he absence of BLM decreases the efficiency of end joining by NHEJ, accompanied by an increase in error-prone events, especially long-range deletions (>200nt). This suggests that BLM protects against extensive resection during alternative NHEJ. Furthermore, BLM is implicated in the protection against CtIP-dependent resection at the initiation of HR. In conclusion, our results show a major role of BLM in protecting against an excess of resection, mediated by the MRN cofactor – CtIP. BLM interacts with 53BP1 at sites of damage, in an ATM-dependent manner, in order to regulate the resection process and counteract BRCA1 activity. This underlines the novel role of BLM in the protection against resection and favouring gene conversion events without crossing-over, which is substantial for maintaining genomic integrity.

In humans, Bloom’s Syndrome is caused by a mutation of the RecQ helicase BLM. Patients with Bloom’s Syndrome exhibit a high amount of genomic instability which results in a high incidence of cancer. Though Bloom’s Syndrome has been intensively studied, there are still many questions about the function of BLM which need to be answered. While it is clear that loss of BLM increases genomic instability, the other effects of genomic instability on the organism aside from cancer such as a potential effect on aging, have yet to be elucidated. In Chapter II, I identify new phenotypes in the C. elegans ortholog of BLM, him-6. him-6 mutants have an increased rate of cell death, a mortal germ line phenotype, and an increased rate of mutations. Upon further examination of the mutator phenotype, it was determined that the increased rate of mutations was caused by small insertions and deletions. The mutator phenotype identified in him-6 mutants closely mimics the cellular phenotype seen in Bloom’s Syndrome cells. This indicates that HIM-6 may behave in a similar fashion to BLM. In addition to the mutator phenotype, it was found that loss of him-6causes a shortened life span. This may provide evidence that there is a link between genomic stability and aging. In Chapter III, I identify a new role for the transcription factor DAF-16. DAF-16 in C. elegans has been intensively studied and regulates a wide variety of pathways. In this chapter, I demonstrate via the well established unc-93 assay that loss of daf-16 causes a subtle mutator phenotype in C. elegans. This indicates that DAF-16 may play a role in suppression of spontaneous mutation. When I examined other classic genomic instability phenotypes, I found at 25°C, the number of progeny in the DAF-16 mutants was significantly reduced compared to wild type worms. Additionally, I demonstrate daf-16(mu86)has a cell death defect. This study identifies several new phenotypes caused by a loss of him-6. These phenotypes provide further evidence that loss of him-6 causes genomic instability. In addition, this study also demonstrates that him-6 has a shortened life span which may be due to genomic instability. Secondly, this study identifies a new role for DAF-16 in preventing the occurrence of spontaneous mutations. This may indicate a novel function for DAF-16 in maintaining genomic stability.

Type I interferons (IFNs) as part of the innate immune system have an outstanding importance as antiviral defense cytokines that stimulate innate and adaptive immune responses. Upon sensing of pattern recognition particles (PRPs) such as nucleic acids, IFN secretion is activated and induces the expression of interferon stimulated genes (ISGs). Uncontrolled constitutive activation of the type I IFN system can lead to autoinflammation and autoimmunity, which is observed in autoimmune disorders such as systemic lupus erythematodes and in monogenic interferonopathies. They are caused by mutations in genes which are involved in sensing or metabolism of intracellular nucleic acids and DNA repair. Many authors described mechanisms of type I IFN secretion upon increased DNA damage, including the formation of micronuclei, cytosolic chromatin fragments and destabilization of DNA binding proteins. Hereditary cutaneous DNA damage syndromes, which are caused by mutations in proteins of the DNA repair, share laboratory and clinical features also seen in autoimmune disorders and interferonopathies; hence a potential role of DNA-damage-induced type I IFN secretion seems likely. Here, we aim to summarize possible mechanisms of IFN induction in cutaneous DNA damage syndromes with defects in the DNA double-strand repair and nucleotide excision repair. We review recent publications referring to Ataxia teleangiectasia, Bloom syndrome, Rothmund–Thomson syndrome, Werner syndrome, Huriez syndrome, and Xeroderma pigmentosum. Furthermore, we aim to discuss the role of type I IFN in cancer and these syndromes.

A síndrome de Bloom (SB) é uma síndrome de instabilidade cromossômica rara, transmitida por herança autossômica recessiva. Caracteriza-se por deficiência de crescimento pré e pós-natal, microcefalia, hipoplasia malar, eritema telangiectásico em face e comprometimento do sistema imunológico, entre outras manifestações clínicas. Os pacientes com SB apresentam predisposição aumentada para o desenvolvimento de neoplasias em idade precoce, sendo esta, a principal causa de óbito. Ao estudo citogenético observa-se aumento de quebras cromossômicas espontâneas e trocas entre cromátides irmãs (TCI), que são utilizadas como marcador diagnóstico para a SB. Além disso, a literatura mostra que a maioria dos pacientes também apresenta mutações no gene BLM, que estão relacionadas a defeitos no mecanismo de reparo do DNA. No entanto, os mecanismos fisiopatológicos não são completamente compreendidos. Nesse sentido estudamos o transcriptoma de duas pacientes portadoras da síndrome de Bloom e de três controles utilizando a metodologia RNA-seq (Illumina, Inc., San Diego, CA). A análise de expressão diferencial revelou 216 genes diferencialmente expressos relacionados a vias relacionadas à resposta imune como replicação negativa da regulação da replicação do genoma viral, regulação positiva da proliferação de células B, via de sinalização mediada por interferon gama, ativação de células B, resposta a vírus, resposta imune adaptativa e processo efetor imune, e nenhuma diferença da expressão em genes de reparo de DNA. Concomitantemente, observamos a hiperexpressão do gene BLM para ambas as pacientes, contribuindo para a desestabilização de genes envolvidos em vias imunológicas, fenômeno também observado em alguns tumores. Dessa forma, sugerimos que a combinação de defeitos de proliferação linfocitária e defeitos de sinalização celular somados a outros, como perda celular e expressão alterada do gene BLM, podem contribuir diretamente para as principais características observadas na síndrome de Bloom, como a deficiência de crescimento e o elevado risco de câncer. Futuramente, o estudo do transcriptoma, aplicado a outros portadores da SB e outras síndromes de instabilidade, possibilitará uma análise mais acurada das interações gênicas relevantes para a desestabilização do genoma / Bloom Syndrome (BS) is a rare chromosome instability syndrome, with recessive autosomal inheritance. The main clinical manifestations are pre and postnatal growth deficiency, microcephaly, malar hypoplasia, telangiectasic facial erythema and compromised immune system, among others. Patients with BS present increased risk to the development of neoplasias at an early age, which is the main cause of death. Cytogenetic test is used as a diagnostic marker for BS since the patient\'s cells present increase in spontaneous chromosomal breaks and sister chromatid exchange (SCE). In addition, the literature reveals that most patients also present mutations in the BLM gene, which are related to defects in the DNA repair mechanism; however, it is still not completely understood. In this sense, we studied the transcriptome of two patients with Bloom\'s syndrome and three controls using the RNA-seq methodology (Illumina, Inc., San Diego, CA). Differential expression analysis revealed 216 differentially expressed genes related to immunological pathways such as: negative replication of the regulation of the viral genome replication, positive regulation of B cells proliferation, gama-interferon mediated signalization pathway, B cells activation, virus response, adaptive immune response and immune effector process, and absence of difference of DNA repair genes expression. At the same time, we observed the hyperexpression of the BLM gene for both patients contributing for the destabilization of genes involved in immunological pathways, a phenomenon also observed in some tumors. Thus, we suggest that the combination of lymphoid proliferation defects and cell signaling defects added to others such as cell loss and altered expression of the BLM gene may contribute directly to the main characteristics observed in Bloom\'s syndrome, such as growth failure and high risk of cancer. In the future, the study of the transcriptome applied to other BS carriers and other instability syndromes, will allow a more accurate analysis of the relevant gene interactions to the destabilization of the genome

A síndrome de Bloom (SB) é uma síndrome de instabilidade cromossômica rara, transmitida por herança autossômica recessiva. Caracteriza-se por deficiência de crescimento pré e pós-natal, microcefalia, hipoplasia malar, eritema telangiectásico em face e comprometimento do sistema imunológico, entre outras manifestações clínicas. Os pacientes com SB apresentam predisposição aumentada para o desenvolvimento de neoplasias em idade precoce, sendo esta, a principal causa de óbito. Ao estudo citogenético observa-se aumento de quebras cromossômicas espontâneas e trocas entre cromátides irmãs (TCI), que são utilizadas como marcador diagnóstico para a SB. Além disso, a literatura mostra que a maioria dos pacientes também apresenta mutações no gene BLM, que estão relacionadas a defeitos no mecanismo de reparo do DNA. No entanto, os mecanismos fisiopatológicos não são completamente compreendidos. Nesse sentido estudamos o transcriptoma de duas pacientes portadoras da síndrome de Bloom e de três controles utilizando a metodologia RNA-seq (Illumina, Inc., San Diego, CA). A análise de expressão diferencial revelou 216 genes diferencialmente expressos relacionados a vias relacionadas à resposta imune como replicação negativa da regulação da replicação do genoma viral, regulação positiva da proliferação de células B, via de sinalização mediada por interferon gama, ativação de células B, resposta a vírus, resposta imune adaptativa e processo efetor imune, e nenhuma diferença da expressão em genes de reparo de DNA. Concomitantemente, observamos a hiperexpressão do gene BLM para ambas as pacientes, contribuindo para a desestabilização de genes envolvidos em vias imunológicas, fenômeno também observado em alguns tumores. Dessa forma, sugerimos que a combinação de defeitos de proliferação linfocitária e defeitos de sinalização celular somados a outros, como perda celular e expressão alterada do gene BLM, podem contribuir diretamente para as principais características observadas na síndrome de Bloom, como a deficiência de crescimento e o elevado risco de câncer. Futuramente, o estudo do transcriptoma, aplicado a outros portadores da SB e outras síndromes de instabilidade, possibilitará uma análise mais acurada das interações gênicas relevantes para a desestabilização do genoma / Bloom Syndrome (BS) is a rare chromosome instability syndrome, with recessive autosomal inheritance. The main clinical manifestations are pre and postnatal growth deficiency, microcephaly, malar hypoplasia, telangiectasic facial erythema and compromised immune system, among others. Patients with BS present increased risk to the development of neoplasias at an early age, which is the main cause of death. Cytogenetic test is used as a diagnostic marker for BS since the patient\'s cells present increase in spontaneous chromosomal breaks and sister chromatid exchange (SCE). In addition, the literature reveals that most patients also present mutations in the BLM gene, which are related to defects in the DNA repair mechanism; however, it is still not completely understood. In this sense, we studied the transcriptome of two patients with Bloom\'s syndrome and three controls using the RNA-seq methodology (Illumina, Inc., San Diego, CA). Differential expression analysis revealed 216 differentially expressed genes related to immunological pathways such as: negative replication of the regulation of the viral genome replication, positive regulation of B cells proliferation, gama-interferon mediated signalization pathway, B cells activation, virus response, adaptive immune response and immune effector process, and absence of difference of DNA repair genes expression. At the same time, we observed the hyperexpression of the BLM gene for both patients contributing for the destabilization of genes involved in immunological pathways, a phenomenon also observed in some tumors. Thus, we suggest that the combination of lymphoid proliferation defects and cell signaling defects added to others such as cell loss and altered expression of the BLM gene may contribute directly to the main characteristics observed in Bloom\'s syndrome, such as growth failure and high risk of cancer. In the future, the study of the transcriptome applied to other BS carriers and other instability syndromes, will allow a more accurate analysis of the relevant gene interactions to the destabilization of the genome