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Homologous Recombination (HR) is a major double-stranded break (DSB) DNA repair pathway. It utilizes the sister chromatid as a template, thus promoting error-free repair. The initial HR step is DNA end resection with the creation of RPA coated single-stranded DNA (ssDNA) overhangs. This is followed by RAD51 loading onto the resected DNA, triggering strand invasion, and homology search. The BRCA1 protein is fundamental for HR mediated DNA repair, and patients with germline BRCA1 mutations have a high risk of developing cancer. These BRCA1 mutant tumors are HR deficient, and as a result, sensitive to PARP inhibitors (PARPi). BRCA1 likely promotes DNA end resection by displacing 53BP1 from the DSBs and also promotes RAD51 loading by enabling the formation of the larger BRCA1-PALB2-BRCA2 complex trough its coiled-coil region. Although BRCA1 functions in both HR steps, its essential role in each step is unclear, with previous reports showing that RAD51 loading after DNA end resection occurred could be BRCA1 independent. Here, we demonstrated that full HR restoration after activation of end resection mediated by 53BP1 knockout (KO), requires the expression of a BRCA1 protein that retained the coiled-coil region and its protein interactions. We observed that although 53bp1 KO can restore the Mendelian frequencies of a Brca1 null mouse model, mice were still tumor prone, PARPi sensitive, and had low levels of RAD51 γ-irradiation-induced foci (IRIF), suggesting that HR levels were minimal. Moreover, in order to significantly increase PARPi resistance in human cancer cell lines, 53BP1 loss of function (LoF) needs to be associated with the expression of a hypomorphic BRCA1 protein that retained the PALB2 interaction. We also demonstrated that Brca1 compound heterozygosity could rescue the developmental defects observed in Brca1 mutant homozygous mice, as long as the alleles retained complementary HR-related functions. Additionally, these compound heterozygotes mice were not tumor prone and had a normal lifespan, suggesting that HR was restored. In contrast, when the allele combination did not exhibit complementary functions, the developmental defects persisted. Overall, we demonstrated that to fully function in mouse development, tumor suppression, and drug resistance, BRCA1 role is critical in both the DNA end resection and the RAD51 loading HR steps. / Biomedical Sciences

À l'heure actuelle, les deux principaux gènes de susceptibilité au cancer du sein et/ou de l'ovaire ont été identifiés, soient les gènes BRCA1 et BRCA2. Ces deux principaux gènes auraient un rôle important à jouer dans le maintien de l'intégrité du génome. Les porteuses d'un gène BRCA1/2 muté auraient un risque d'être atteintes d'un cancer du sein de 45% à 65% avant l'âge de 70 ans, tandis que cette valeur serait de 11% à 39% pour le cancer de l'ovaire, lorsqu'estimé à partir d'individus non-sélectionnées sur la base de l'histoire familiale. Non seulement, il existe à l'heure actuelle une divergence importante quant aux estimations concernant la contribution relative et l'impact des gènes BRCA1 et BRCA2 dans les cas familiaux de cancer du sein, mais les données provenant des autres populations étudiées ne peuvent être extrapolées à la population canadienne-française, puisque nous sommes en présence d'un effet fondateur. Nous avons démontré que la majorité des familles canadienne-française du Québec à risque élevé de développer un cancer du sein et/ou de l'ovaire porteuses d'une mutation délétères de BRCA1 ou BRCA2 pouvaient être expliquées par la présence de 11 mutations délétères récurrentes et c'est la première étude à déterminer la proportion des familles BRCA1, BRCA2 ou BRCAX dans la population canadienne-française à haut risque. Nous avons aussi démontré que l'utilisation du modèle empirique de Manchester est efficace afin de prédire la probabilité qu'une famille soit porteuse d'une mutation dans cette même population. De plus, suite à des analyses de type « Southern blot » et « multiplex ligation-dependent probe amplification » (MLPA), la présence de mutations de type réarrangements chromosomiques semble très rare dans notre population puisque si de telles altérations avaient été présente à une fréquence supérieure à 2.2%, nous aurions eu 95% de chance d'en identifier au moins une dans les 135 familles étudiées par ces deux méthodes. Finalement, nous avons mis en évidence un nouveau transcrit du gène BRCA1. Celui-ci est généré par un épissage alternatif créant un nouvel exon, apelé exon 13A, constitué d'un segment de 66 nucléotides. Les résultats de ces différentes études démontrent l'apport qu'ont les mutations délétères de BRCA1 et BRCA2 chez les canadiennes-françaises. Ces éléments de réponse pourraient avoir un impact majeur sur la santé et sur la prise en charge des femmes ou hommes porteurs de telles mutations.

Current sequencing technologies allows researchers unprecedented insight into our biology, and how these biological mechanisms can become distorted and lead to disease. These aberrant mechanisms can be brought about by many causes, but some occur as a result of genetic mutations or external factors through the epigenome. Here, we used our microfluidic technology to profile the epigenome and transcriptome to study such aberrant mechanisms in three different diseases and illnesses: breast cancer, chronic inflammation, and mental illness. We profiled the epigenome of breast tissue from healthy women with the BRCA1 mutation to understand how the mutation may facilitate eventual breast cancer. Epigenomic changes in breast cells suggest that cells in the basal compartment may differentiate into a different cell type, and perhaps become the source of breast cancer. Next, we compared the epigenome and genome of murine immune cells under low-grade inflammation and acute inflammation conditions. We found that low-grade inflammation preferentially utilizes different signaling pathways than in acute inflammation, and this may lead to a non-resolving state. Finally, we analyzed the effect of the maternal immune activation on unborn offspring, and how these changes could cause later mental illness. The insights we made into these diseases may lead to future therapies. / Doctor of Philosophy / Despite advances in medical and scientific research, there is still a dearth of information on how diseases affect the expression of our genes, such as breast cancer, chronic inflammation, and influenza. Mutation in the BRCA1 gene is probably the most well-known mutation that can lead to breast cancer. We know the overarching reason that mutation in BRCA1 can lead to cancer, as BRCA1 is responsible for repairing damage in the DNA, so mutations can compound and create cancerous cells. However, we do not know the exact mechanisms by which this actually happens. Another widespread problem is chronic inflammation, which can promote or lead to diseases such as diabetes, cancer, Alzheimer's, Rheumatoid arthritis, and heart disease. In addition, there are many causes of chronic inflammation that many people have experienced at some point in time, including stress, insomnia, being sedentary, poor eating habits, and obesity. Despite this, we still do not fully understand why chronic inflammation differs from normal inflammation, which is a healthy process, or why it does not resolve. There are also other connections that are surprising, and many are not aware of. If a pregnant woman gets the flu during her second trimester, her baby has much higher odds of developing schizophrenia later in its lifetime. Given the prevalence of the flu, there is a very real chance that an expecting mother will be infected during her pregnancy.

Cette étude décrit les perceptions des bénéfices, des risques et des limites des tests BRCA1/2 de 650 femmes participant à un programme de recherche clinique intégré incluant des tests de prédisposition génétique au cancer du sein. Ces perceptions sont décrites en fonction de l'âge, du statut marital, du statut parental et des antécédents personnels de cancer des participantes. En général, les femmes testées perçoivent que les bénéfices du test sont plus importants que les risques et les limites. Les résultats indiquent que la principale motivation des femmes testées est le désir de faire progresser la science et le principal risque perçu est la peur que le résultat du test perturbe la famille. Les implications du résultat du test pour la surveillance et la prévention du cancer sont particulièrement importantes pour les femmes plus jeunes et celles n'ayant jamais fait de cancer.

La susceptibilité d'un individu à développer un cancer du sein est le résultat d'une interaction complexe de facteurs reliés au style de vie, à l'histoire reproductive et aux déterminants génétiques propres à chaque individu. Jusqu'à présent, un nombre limité de gènes ont été impliqués dans une telle susceptibilité. Il est présentement estimé que des mutations et variations de l'ensemble des gènes de susceptibilité connus (par exemple BRCA1, BRCA2, TP53, STK11, PTEN, ATM, PALB2, CHEK2, BR1P1 et les alleles de faible penetrance identifiés récemment) ne seraient responsables que d'un maximum de 30% des cas de cancers clairement familiaux. Dans cette thèse, la contribution de certains gènes a été investiguée dans une cohorte de femmes provenant de la population canadienne française et présentant des évidences claires de l'implication forte de facteurs génétiques de susceptibilité non reliés à BRCA1 ou BRCA2. Notre étude concerne les gènes candidats ZBRK1 (Zinc finger and BRCA1-interacting protein with KRAB domain 1), GADD45A (Growth arrest and DNA-damage-inducible alpha) et NBS1 (Nijmegen breakage syndrome 1). Notre analyse de ZNF350/ZBRK1 a permis de mettre en évidence trois haplotypes modulant de façon significative le risque de cancer du sein dans notre population. Parmi ceux-ci, deux pourraient être associés à un effet protecteur (p=0.01135 et p=0.00268) alors qu'un autre haplotype est lié à une augmentation du risque (p=0.00143). Dans le cas de GADD45A, nous avons identifié un haplotype commun démontrant une fréquence plus élevée dans le groupe contrôle, bien que cette association soit faible. En ce qui concerne NBN, le variant du promoteur c.-242-l lOdelAGTA est significativement surreprésenté dans notre groupe de femmes atteintes. Des essais de type gène luciférase rapporteur n'ont pas démontré de variation d'expression dans la lignée cellulaire de cancer du sein MCF-7, mais ont indiqué une réduction de l'expression dans les lignées cellulaires HEK293 et LNCaP. Ces résultats indiquent qu'il est possible que des variations de ces gènes, bien que n'étant pas très fréquentes, soient impliquées dans la susceptibilité au cancer du sein dans notre population et des études plus approfondies sur de grandes cohortes seront nécessaires afin de confirmer ou infirmer nos résultats.

INTRODUÇÃO: O câncer constitui-se na principal causa de mortalidade entre indivíduos de 45 a 84 anos, configurando-se em um dos principais problemas de saúde pública dos países em desenvolvimento. As reações de hipersensibilidade aos quimioterápicos têm aumentado, impedindo muitas vezes a utilização de terapias de primeira linha no tratamento de neoplasias primárias ou recidivantes. O procedimento de dessensibilização é uma abordagem alternativa, por meio do qual o paciente passa a tolerar a medicação que antes desencadeava reações potencialmente letais. Quimioterápicos do grupo das platinas são exemplos de drogas passíveis de readministração por meio do processo de dessensibilização, no entanto faltam biomarcadores preditivos de reações durante o procedimento. OBJETIVOS: O objetivo principal do estudo foi avaliar o papel do teste de ativação de basófilos (BAT) como biomarcador para reações de hipersensibilidade ocorridas durante a dessensibilização em pacientes alérgicas às platinas. Como objetivo secundário, avaliou-se a prevalência e o impacto da mutação dos genes BRCA 1 e 2 em pacientes com hipersensibilidade imediata à carboplatina submetidas à dessensibilização. MÉTODOS: Padronizou-se o BAT, com análise da expressão de CD63 e CD203c na superfície de basófilos de pacientes com hipersensibilidade imediata às platinas submetidas à dessensibilização. Foram realizados BATs em 15 pacientes portadoras de neoplasias malignas submetidas a 27 dessensibilizações devido à anafilaxia a quimioterápico do grupo das platinas e em 12 indivíduos de dois grupos controle (Grupo 1: seis pacientes tolerantes às platinas e Grupo 2: seis voluntários sadios que nunca foram expostos às platinas). Os resultados dos BATs foram comparados entre os três grupos. Correlacionou-se o BAT com a ocorrência ou não de reação durante a dessensibilização e com os níveis de triptase sérica. Para análise da prevalência e impacto da mutação dos genes BRCA 1 e 2 nas dessensibilizações, realizou-se análise retrospectiva de prontuários de 138 portadoras de neoplasias malignas ginecológicas submetidas à dessensibilização à carboplatina. RESULTADOS: O BAT foi positivo em 11 das 15 pacientes alérgicas (n= 11; 73,3%), com aumento de expressão de CD203c e CD63 em 11 (73,3%) e 6 (40%) pacientes, respectivamente. Todos os participantes dos grupos controles apresentaram testes negativos. Maior expressão de CD63 foi observada em pacientes com reações iniciais mais graves. O BAT foi positivo em 92,3% das reações ocorridas durante as dessensibilizações (n=12/13), sendo positivo em todas as reações que apresentaram aumento concomitante de triptase sérica (n=5). Com relação à mutação dos genes BRCA 1 e 2, sua prevalência foi de 34% nas pacientes com hipersensibilidade às platinas (n=47/138), sendo que 51% das portadoras reagiram durante a dessensibilização. CONCLUSÕES: O BAT positivo, com aumento da expressão de CD63 e/ou CD203c na superfície do basófilo, identificou pacientes alérgicos às platinas com especificidade de 100% e sensibilidade de 73,3%. O BAT e a mutação dos genes BRCA 1 e 2 identificaram pacientes mais propensos a reagir durante o procedimento de dessensibilização. A utilização de biomarcadores preditores de reações durante a dessensibilização aos quimioterápicos do grupo das platinas pode aumentar a segurança do procedimento e auxiliar na manutenção do esquema quimioterápico de primeira linha do paciente / INTRODUCTION: Cancer is the leading cause of death in the age group of 45 to 84 years, and one of the main public health issues in developing nations. Hypersensitivity reactions to chemotherapeutic agents have been increasing, sometimes hindering the use of first-line therapies in the treatment of primary or relapsed tumors. Rapid drug desensitization (RDD) is an alternative approach, through which a patient becomes tolerant to the medication that once triggered a potentially lethal hypersensitivity reaction. Platinum-based compounds are examples of drugs that can be readministered through the desensitization procedure, but currently there are no known biomarkers that could help predict reactions during RDD. OBJECTIVES: The main goal of our study was to assess the basophil activation test (BAT) as a biomarker of breakthrough reactions occurred during RDD in patients allergic to platinum-based agents. As a secondary goal, we evaluated the prevalence and impact of the BRCA 1/2 mutation in carboplatin-allergic patients undergoing RDD. METHODS: We standardized the BAT by evaluating CD63 and CD203c expressions on the basophils of patients with immediate hypersensitivity reactions to platinum-based agents undergoing RDD. We analyzed BATs of 15 patients with malignant neoplasms who had undergone 27 RDD procedures due to anaphylaxis to platinum-based agents, and of 12 control subjects (Group 1: six patients tolerant to platinum-based agents, and Group 2: six healthy volunteers who had never been exposed to platinum-based agents). BAT results were compared among the three groups. We correlated BAT results with the occurrence of breakthrough reactions during RDD and with serum tryptase levels. To conduct the analysis of the BRCA 1/2 mutation prevalence and its impact on RDD, a retrospective review of 138 medical records of patients with gynecological malignancies who underwent RDD to carboplatin was performed. RESULTS: BAT was positive in 11/15 allergic patients (73.3%), with increased expression of CD203c and CD63 in 11 (73.3%) and 6 (40%) patients, respectively. All control subjects presented negative BATs. A higher CD63 expression was observed in patients with severe initial reactions. BAT was positive in 92.3% of the breakthrough reactions occurred during RDD (n=12/13), and in all reactions with concomitant increased tryptase levels (n=5). Regarding the BRCA1/2 mutation, its prevalence was 34% in patients allergic to platinum-based agents (n=47/138), and 51% of the mutation carriers had breakthrough reactions during RDD. CONCLUSIONS: A positive BAT, with an increased expression of CD63 and/or CD203c, identified patients allergic to platinum-based agents with a specificity of 100% and a sensitivity of 73.3%. The BAT and the BRCA 1/2 mutation helped identify patients at risk of breakthrough reactions during RDD. The use of predictive biomarkers of breakthrough reactions during RDD to platinum-based agents might enhance RDD safety and help maintain a patient`s first-line treatment

INTRODUÇÃO: Diversas sociedades profissionais recomendam a realização de testes genéticos para mulheres que desenvolveram câncer de ovário, a fim de identificar portadores de mutação germinativa em genes BRCA1/2 e oferecer terapia redutora de risco. OBJETIVO: O objetivo deste estudo foi realizar análise de custo-efetividade de programa para diagnóstico de mutação germinativa em genes BRCA1/2 e de estratégias preventivas para pacientes com o diagnóstico de câncer de ovário e seus familiares de primeiro grau. METODOLOGIA: O estudo realizou análise de custo-efetividade mediante desenvolvimento de modelo de decisão de Markov e perspectiva do Sistema Único de Saúde. As estratégias comparadas refletiram a adoção de teste genético e estratégias preventivas para pacientes e familiares ou o acompanhamento proposto atualmente. A razão de custo-efetividade incremental foi expressa em termos de custo por caso evitado de neoplasia maligna. A análise de sensibilidade foi realizada de forma determinística univariada. RESULTADOS: Demonstrou-se incremento em efetividade e em custos com a realização de testes genéticos e a adoção de medidas profiláticas para pacientes e familiares. A razão de custo-efetividade incremental foi calculada em R$ 14.224,40 e em R$ 908,58, respectivamente, por caso evitado em pacientes com o diagnóstico prévio de câncer de ovário e em seus familiares de primeiro grau. Estes valores foram considerados inferiores ao limiar de custo-efetividade selecionado no estudo (de R$ 7.543,50 a R$ 23.786,70). DISCUSSÃO: O programa analisado pode ser considerado como estratégia custo-efetiva para a realidade nacional, sobretudo no que tange aos familiares de primeiro grau de pacientes com o diagnóstico de câncer de ovário. Outras publicações demonstraram conclusões similares para o tema em diversos países. CONCLUSÃO: Um possível desdobramento deste trabalho poderia ser representado pela realização de uma análise de impacto orçamentário da incorporação do programa como política de saúde no país / INTRODUCTION: Several professional societies recommend performing genetic tests for women who have developed ovarian cancer in order to identify BRCA1/2 germline-mutation carriers and offer risk-reducing therapy. OBJECTIVE: The objective of this study was to perform a cost-effectiveness analysis of a BRCA1/2 germline mutation diagnosis program and preventive strategies for patients diagnosed with ovarian cancer and their first degree relatives. METHODS: The study performed a cost-effectiveness analysis through the development of a Markov decision model and the perspective of the Unified Health System. The compared strategies reflected the adoption of genetic testing and preventive strategies for patients and their relatives or the usual follow-up. The incremental cost-effectiveness ratio was expressed in terms of cost per avoided case of cancer. Sensitivity analysis was performed in a univariate and deterministic manner. RESULTS: There has been an increase in effectiveness and in costs with genetic testing and the adoption of prophylactic measures for patients and their relatives. The incremental cost-effectiveness ratio was calculated at R$ 14,224.40 and R$ 908.58, respectively, for avoided cases in patients with prior diagnosis of ovarian cancer and their first-degree relatives. These values were considered lower than the cost-effectiveness threshold selected in the study (from R$ 7,543.50 to R$ 23,786.70). DISCUSSION: The analyzed program can be considered as a cost-effective strategy for the national reality, especially in relation to the first-degree relatives of patients with ovarian cancer. Other publications have shown similar conclusions for the subject in several countries. CONCLUSION: A possible development of this work could be represented by a budget impact analysis of the incorporation of the program as health policy in Brazil

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