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Análise Integrativa De Perfis Transcricionais De Pacientes Com UNIVERSIDADE DE SÃO PAULO FACULDADE DE MEDICINA DE RIBEIRÃO PRETO PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA ADRIANE FEIJÓ EVANGELISTA Análise integrativa de perfis transcricionais de pacientes com diabetes mellitus tipo 1, tipo 2 e gestacional, comparando-os com manifestações demográficas, clínicas, laboratoriais, fisiopatológicas e terapêuticas Ribeirão Preto – 2012 ADRIANE FEIJÓ EVANGELISTA Análise integrativa de perfis transcricionais de pacientes com diabetes mellitus tipo 1, tipo 2 e gestacional, comparando-os com manifestações demográficas, clínicas, laboratoriais, fisiopatológicas e terapêuticas Tese apresentada à Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo para obtenção do título de Doutor em Ciências. Área de Concentração: Genética Orientador: Prof. Dr. Eduardo Antonio Donadi Co-orientador: Prof. Dr. Geraldo A. S. Passos Ribeirão Preto – 2012 AUTORIZO A REPRODUÇÃO E DIVULGAÇÃO TOTAL OU PARCIAL DESTE TRABALHO, POR QUALQUER MEIO CONVENCIONAL OU ELETRÔNICO, PARA FINS DE ESTUDO E PESQUISA, DESDE QUE CITADA A FONTE. FICHA CATALOGRÁFICA Evangelista, Adriane Feijó Análise integrativa de perfis transcricionais de pacientes com diabetes mellitus tipo 1, tipo 2 e gestacional, comparando-os com manifestações demográficas, clínicas, laboratoriais, fisiopatológicas e terapêuticas. Ribeirão Preto, 2012 192p. Tese de Doutorado apresentada à Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo. Área de Concentração: Genética. Orientador: Donadi, Eduardo Antonio Co-orientador: Passos, Geraldo A. 1. Expressão gênica – microarrays 2. Análise bioinformática por module maps 3. Diabetes mellitus tipo 1 4. Diabetes mellitus tipo 2 5. Diabetes mellitus gestacional FOLHA DE APROVAÇÃO ADRIANE FEIJÓ EVANGELISTA Análise integrativa de perfis transcricionais de pacientes com diabetes mellitus tipo 1, tipo 2 e gestacional, comparando-os com manifestações demográficas, clínicas, laboratoriais, fisiopatológicas e terapêuticas. Tese apresentada à Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo para obtenção do título de Doutor em Ciências. Área de Concentração: Genética Aprovado em: Banca Examinadora Prof. Dr. _______________________________________________________________ Instituição:_______________________Assinatura:_____________________________ Prof. Dr. _______________________________________________________________ Instituição:_______________________Assinatura:_____________________________ Prof. Dr. _______________________________________________________________ Instituição:_______________________Assinatura:_____________________________ Prof. Dr. _______________________________________________________________ Instituição:_______________________Assinatura:_____________________________ Prof. Dr. _______________________________________________________________ Instituição:_______________________Assinatura:_____________________________ Dedico especialmente este trabalho Aos meus pais Laercio e Rita Ao meu irmão Renato Ao meu namorado Eduardo AGRADECIMENTOS Ao meu orientador Prof. Dr. Eduardo Antonio Donadi pela oportunidade de participar do seu grupo de pesquisa, pelo apoio científico e pessoal, pelo exemplo a ser seguido. Ao meu co-orientador no Brasil Prof. Dr. Geraldo Aleixo da Silva Passos Jr. e ao meu supervisor estrangeiro na França Prof. Dr. Denis Puthier pela imensa contribuição neste trabalho, e pela formação científica. Aos pesquisadores participante do projeto temático FAPESP, ao qual este trabalho está inserido. A Prof. Dra. Elza Tiemi Sakamoto-Hojo, pela contribuição científica e correção do manuscrito para publicação. Ao prof. Dr. Milton Cesar Foss e à prof. Dra. Maria Cristina Foss-Freitas pela orientação clínica e seleção dos pacientes. À Dra. Diane Meyre Rassi pela participação na etapa da seleção e coleta das amostras. À equipe francesa. À Prof. Dra. Catherine Nguyen, diretora da unidade INSERM-U928, pelo apoio e autorização da utilização das dependências e treinamento. Ao Prof. Dr. Pascal Rihet pelas discussões e sugestões de análises, de grande valia neste trabalho. Aos Prof. Dr. Carl Hermann e Prof. Dr. Sammuel Granjeau pelas sugestões e ensinamentos em programação em linguagens R e PERL. Aos técnicos Hèlène Holota, Bèatrice Loriod e, em especial, à Gènèvieve Victorero por todos os ensinamentos. A divisão de endocrinologia do Hospital das Clinicas da Faculdade de Medicina de Ribeirão Preto, em cujas dependências foram obtidas as amostras. Ao laboratório de HLA do Hemocentro da Faculdade de Medicina de Ribeirão Preto, em especial à técnica Neife Deghaide. A todos os funcionários que direta ou indiretamente contribuíram na realização deste trabalho. Aos pacientes, sem os quais esse trabalho não poderia ser realizado. Ao Laboratório de Imunogenética Molecular do Departamento de Genética da Faculdade de Medicina de Ribeirão Preto (FMRP/USP). Agradeço aos colegas Danilo Xavier, Cristhianna Collares, Renata Almeida, Paula Takahashi, Fernanda Manoel-Caetano, Thais Arns, Natália Joanne, Amanda Assis, Flávia Porto, Juliana Massaro, Ernna Domingues, Claudia Macedo, Thais Fornari, Paula Donate, Janaina Dernowsek e Nicole Pezzi. Aos colegas que contribuíram com a minha formação desde o mestrado: Cristina Junta e Márcia Marques. Ao programa de Pós-graduação em Genética, do Departamento de Genética da Faculdade de Medicina de Ribeirão Preto – USP. Agradeço aos professores pela qualidade da formação e aos colegas de pós-graduação. Agradeço à secretaria do departamento, em especial às secretárias Susie, Silvia e Maria Aparecida pela eficiência e ajuda todos esses anos. Ao CNPq FAPESP, e FAEPA pelo apoio financeiro necessário para a realização deste trabalho. Aos membros da banca examinadora pela prontidão, contribuições, críticas e sugestões. Aos meus queridos amigos Ana Durvalina Bomtorin, Breno Mello, Ana Rita Batistela e Andressa Morales pelo companheirismo. Aos amigos da França, Miriam Yammine, Elise Yammine, Alberto Marcus, Joanna Bou Saab, Daou Pascale, Sabrina Sanfilippo, Yuska Aguiar e Luciana Lopes pelas discussões científicas e pelos grandes momentos vividos. Aos meus pais Laércio e Rita pelo exemplo de vida e superação. A toda minha família que sempre me apoiou. Ao meu namorado Carlos Eduardo Silva Lazzarini, e à sua família, pelo companheirismo, paciência e incentivo de extrema importância no período de realização deste trabalho, e em todos os momentos. RESUMO Evangelista, A. F. Análise integrativa de perfis transcricionais de pacientes com diabetes mellitus tipo 1, tipo 2 e gestacional, comparando-os com manifestações demográficas, clínicas, laboratoriais, fisiopatológicas e terapêuticas. 2012. 192p. Tese (Doutorado) – Faculdade de Medicina de Ribeirão Preto, 2012. O diabetes mellitus tipo 1 (DM1) tem etiologia autoimune, enquanto o diabetes mellitus tipo 2 (DM2) e o diabetes mellitus gestacional (DMG) são considerados como distúrbios metabólicos. Neste trabalho, foi realizada análise do transcriptoma das células mononucleares do sangue periférico (do inglês, peripheral mononuclear blood cells - PBMCs), obtidas de pacientes com DM1, DM2 e DMG, realizando análises por module maps a fim de comparar características patogênicas e aspectos gerais do tratamento com anotações disponíveis de genes modulados, tais como: a) análises disponíveis a partir de estudos de associação em larga escala (do inglês genome-wide association studies – GWAS); b) genes associados ao diabetes em estudos clássicos de ligação disponíveis em bancos de dados públicos; c) perfis de expressão de células imunológicas fornecidos pelo grupo ImmGen (Immunological Project). Foram feitos microarrays do transcriptoma total da plataforma Agilent (Whole genome one- color Agilent 4x44k) para 56 pacientes (19 DM1, 20 DM2 e 17 DMG). Para a compreensão dos resultados foram aplicados filtros não-informativos e as listas de genes diferencialmente expressos foram obtidas por análise de partição e análise estatística não-paramétrica (rank products), respectivamente. Posteriormente, análises de enriquecimento funcional foram feitas pelo DAVID e os module maps construídos usando a ferramenta Genomica. As análises funcionais contribuíram para discriminar os pacientes a partir de genes envolvidos na inflamação, em especial DM1 e DMG. Os module maps de genes diferencialmente expressos revelaram: a) genes modulados exibiram perfis de transcrição típicos de macrófagos e células dendríticas, b) genes modulados foram associados com genes previamente descritos como genes de complicação ao diabetes a partir de estudos de ligação e de meta-análises; c) a duração da doença, obesidade, número de gestações, níveis de glicose sérica e uso de medicações, tais como metformina, influenciaram a expressão gênica em pelo menos um tipo de diabetes. Esse é o primeiro estudo de module maps mostrando a influência de padrões epidemiológicos, clínicos, laboratoriais, imunopatogênicos e de tratamento na modulação dos perfis transcricionais em pacientes com os três tipos clássicos de diabetes: DM1, DM2 e DMG. Palavras-chave: Expressão gênica, microarrays, bioinformática, module maps, diabetes mellitus tipo 1, diabetes mellitus tipo 2, diabetes mellitus gestacional. ABSTRACT Evangelista, A. F. Integrative analysis of transcriptional profiles in type 1, type 2 and gestational diabetes mellitus, compared with demographic, clinical, laboratory, physiopathology and therapeutic manifestations. 2012. 192p. Thesis (PhD Degree) – Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil, 2012. Type 1 diabetes (T1D) is an autoimmune disease
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