TIAGO TAIETE
AVALIAÇÃO DE ALTERAÇÕES GENÉTICAS EM PACIENTES
COM PERIODONTITE AGRESSIVA NA POPULAÇÃO BRASILEIRA
Piracicaba 2017
UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE ODONTOLOGIA DE PIRACICABA
EVALUATION OF GENETIC ALTERATIONS IN PATIENTS WITH
AGGRESSIVE PERIODONTITIS IN THE BRAZILIAN POPULATION
AVALIAÇÃO DE ALTERAÇÕES GENÉTICAS EM PACIENTES COM
PERIODONTITE AGRESSIVA NA POPULAÇÃO BRASILEIRA
Piracicaba
2017
EVALUATION OF GENETIC ALTERATIONS IN PATIENTS WITH
AGGRESSIVE PERIODONTITIS IN THE BRAZILIAN POPULATION
TIAGO TAIETE
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos para a obtenção do título de Doutor em Clínica Odontológica, na Área de Periodontia.
Orientador: Prof. Dr. Renato Corrêa Viana Casarin
ESTE EXEMPLAR CORRESPONDE À
VERSÃO FINAL DA TESE DEFENDIDA PELO ALUNO Tiago Taiete E ORIENTADA PELO PROF. DR. Renato Corrêa Viana Casarin
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requeriments for the degree of Doctor in Clinical Dentistry, in Periodontics area.
DEDICATÓRIA
Aos meus pais Valéria Aparecida Giacomini Taiete e Orlando Taiete, minha eterna gratidão pelo amor incondicional que sempre tiveram comigo. Estiveram presentes em todas as etapas de minha vida, me apoiando e incentivando. Ensinaram-me com seus exemplos a ter responsabilidade, tolerância e honestidade.
Ao meu irmão Ricardo Taiete, grande amigo e exemplo de dedicação e perseverança.
À Maria Alice Gatti Palma, por todo amor, carinho, compreensão e incentivo no meu crescimento profissional e pessoal. Dedico a você esta tese e o futuro.
AGRADECIMENTOS
À Deus, por sua presença constante, ajudando-me a superar cada obstáculo da minha vida.
À Universidade Estadual de Campinas, na pessoa do Magnífico Reitor Prof. Dr. José Tadeu Jorge.
À Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas, nas pessoas do Diretor Prof. Dr. Guilherme Elias Pessanha Henriques e do Diretor Associado Prof. Dr. Francisco Haiter Neto.
À Profa. Dra. Cinthia Pereira Machado Tabchoury, Coordenadora dos Cursos de Pós-graduação, e à Profa. Dra. Karina Gonzales Silvério Ruiz, Coordenadora do Curso de Pós-Graduação em Clínica Odontológica.
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Processo 140967/2013-3) pelo apoio ao trabalho em forma de bolsa de doutorado.
Ao Prof. Dr. Renato Corrêa Viana Casarin, meu amigo e orientador. Exemplo de profissional dedicado, com caráter e paciente. Obrigado por me ensinar a fazer ciência com tanto amor e entusiasmo, a descomplicar quando tudo parece impossível. Serei eternamente grato por todo ensinamento, ajuda e por sempre estar disposto a ouvir minhas dúvidas e anseios. Obrigado por permitir meu crescimento profissional.
Ao Prof. Dr. Marcio Zaffalon Casati, por ser fundamental na minha carreira acadêmica, por acreditar no meu potencial e me ensinar muito sobre periodontia, ciência e a carreira acadêmica. Obrigado por me aceitar no estágio, especialização,
no mestrado, doutorado e por me incluir nas suas pesquisas. Aprendi muito com seu exemplo a ter dedicação e agilidade.
Aos professores da área de periodontia, Prof. Dr. Antônio Wilson Sallum, Prof. Dr. Enilson Antônio Sallum, Prof. Dr. Francisco Humberto Nociti Júnior, Profa. Dra. Karina Gonzales Silvério Ruiz, Prof. Dr. Marcio Zaffalon Casati e Prof. Dr. Renato Corrêa Viana Casarin pelos conhecimentos compartilhados, apoio, disponibilidade e atenção dispensada a mim. Obrigado por permitirem a minha participação nesse programa de pós-graduação de excelência.
Ao prof. Dr. Ricardo Della Coletta por seu auxílio fundamental na execução das análises que compõe minha tese de doutorado. Obrigado pela paciência e disponibilidade em ensinar e por fornecer informações e os reagentes da reta final do meu doutorado.
Às professoras e amigas Mônica Grazieli Correa e Ana Paula Oliveira Giorgetti Bossolan, da Universidade Paulista, por me ajudarem com as coletas de células epiteliais dos pacientes de periodontite agressiva.
Ao professor e amigo Mauro Pedrine Santamaria da FOSJC-UNESP pela cooperação fundamental para a execução deste trabalho, permitindo acesso aos seus pacientes. Agradeço por me ensinar periodontia durante a especialização e mestrado.
À Cassia Fernandes Araújo e à Naira Maria Rebelato Bechara Andere da FSJC-UNESP por disporem do tempo de vocês para me auxiliarem na coleta dos pacientes com periodontite agressiva.
À Isabela Lima França Grohmann e à Manuela Rocha dos Santos por me ajudarem nas coletas dos pacientes com periodontite crônica.
À profa. Dra. Denise Carleto Andia pelos ensinamentos, desde a aula de tecido ósseo na graduação até meu doutorado, e por sua contribuição na execução e análise do exoma.
À Dra. Luciana Souto Mofatto pela sua dedicação na análise de bioinformática deste trabalho. Obrigado pela paciência em executar diversas análises e por me ensinar um pouco de bioinformática.
À profa. Dra. Luciane Martins por sempre me ajudar e ensinar, por sempre fornecer informações importantes para a execução das análises laboratoriais, e por sua disposição e entusiasmo na execução das análises in silico deste trabalho.
Aos professores que participaram da banca de qualificação deste trabalho: Denise Carleto Andia, Luciane Martins, Cristiane Ribeiro Salmon e Luciana Souto Mofatto (suplente), pelas dicas e comentários pertinentes para a redação desta tese.
Aos professores que participaram da banca de defesa deste trabalho de doutorado: Renato Corrêa Viana Casarin, Marcio Zaffalon Casati, Ricardo Della Coletta, Letícia Helena Theodoro, Raquel Mantuaneli Scarel Caminaga, Mauro Pedrine Santamaria (suplente), Cristina Cunha Villar (suplente) e Enilson Antonio Sallum (suplente).
À profa. Dra. Cristiane Ribeiro Salmon por ser um exemplo profissional, por me ajudar e ensinar tanto durante a pós-graduação.
Ao Hugo Felipe do Vale, grande amigo e exemplo profissional, obrigado pela amizade e por todo o incentivo para me tornar um periodontista e pesquisador melhor.
Ao Lucas Alves Moura e Lucas Araújo Queiroz, pela amizade e paciência durante a especialização.
À Maria Fernanda Santos Peres, por me acolher durante o estágio, ensinar a prática clínica de periodontia quando eu estava começando e permitir que eu auxiliasse sua pesquisa.
Aos professores Dr. Reginaldo Bruno Gonçalves e Dra. Regianne Umeko Kamiya, da área de Microbiologia e Imunologia, meus orientadores na iniciação científica. Pela oportunidade de aprender e vivenciar a ciência, o que facilitou minha caminhada durante a pós-graduação.
À Regina Célia Corrêa Caetano da Silva, Mariana Piovezan Fugolin e Eliete Ferreira Lima Marim que sempre estiveram dispostas a me ajudar. Obrigado pela paciência, amizade e bom-humor no dia a dia.
Aos meus amigos desde a graduação Alexsandra Shizue Iwamoto, Katia Borges Batista, Larissa Bortoletto Miyata, Marcos Jaquinta Wood, Miquéias de Oliveira Lima Fernandes, Pedro Augusto Thiene Leme, Rivaldo Carneiro Firmino Filho, Rodolfo Alberto Pires de Camargo, Vinícius Brito Silva, Vinícius Henrique Alves Ferreira e Vinícius Luiz Rodrigues Podadera. As pessoas aparecem na nossa vida por acaso, mas não é por acaso que elas permanecem. Obrigado pela amizade, apoio e momentos de descontração nesses 11 anos de convívio.
Aos amigos e colegas da periodontia das turmas antigas: Ana Paula Giorgetti Bossolan, Ana Regina Oliveira Moreira, Bruna Rabelo Amorim, Camila Camarinha da Silva Cirino, Cristiane Ribeiro Salmon, Hugo Felipe do Vale, Isabela
Lima França, Lucas Araújo Queiroz, Lucas Alves Moura, Luciane Martins, Mabelle de Freitas Monteiro, Mayra Laino Albiero, Mauro Pedrine Santamaria, Miki Taketomi Saito, Mirella Lindoso Gomes Campos, Mônica Grazieli Corrêa, Tiago Tarbes Vianna e Viviene Santana Barbosa. Obrigado pelos bons momentos vividos, trocas de informações e incontáveis ajudas.
Aos amigos e colegas da periodontia da turma nova: Amanda Bandeira de Almeida, Ana Lívia Fileto Santana, Elis Janaina Lira dos Santos, Fernanda Félix Cordeiro Dias, Guilherme Henrique Costa Oliveira, João Paulo Menck Sangiorgio, Manuela Rocha dos Santos, Marcela di Moura Barbosa, Rafaela Videira Clima da Silva, Rahyza Inácio Freire de Assis, Samira Salmeron e Thiago Ozi Bueno.
À Maria Alice Gatti Palma, Isabela Lima França, Miki Taketomi Saito, Mayra Laino Albiero, Lucas Alves Moura e João Paulo Menck Sangiorgio. A pós-graduação seria mais chata sem vocês.
Às alunas de iniciação científica Camila Schmitd Stolf e Paloma do Nascimento, obrigado pela ajuda nas atividades laboratoriais e por permitirem meu início como orientador de pesquisas.
Aos amigos Fernanda Félix Cordeiro Dias, Thiago Ozi Bueno e Tiago Tarbes Vianna pelo trabalho em conjunto e aceitarem minhas ideias e ajuda.
À Fernanda Félix Cordeiro Dias, por sua vontade de aprender. Tenho muito orgulho de ter participado do seu desenvolvimento acadêmico.
À família da Maria Alice, em especial aos meus sogros, Ana Maria Gatti Palma e Carlos Eduardo Palma, por me receberem muito bem, por me apoiarem, e por serem exemplos de pessoas boas e honestas.
Aos meus avós, Zulmira Parazzi Giacomini e Arlindo Giacomini, que sempre acreditaram em mim, por toda alegria em nossa convivência e por me ensinarem com seus exemplos o sentido da vida.
Ao Ricardo e aos meus pais Orlando e Valéria, serei eternamente grato por acreditarem e lutarem por mim, dando toda a estrutura financeira e psicológica para que meus sonhos se tornassem realidades. Obrigado por me ensinarem com seus exemplos a batalhar para alcançar meus objetivos, a ser responsável, dedicado, educado e ético no trabalho e nas relações pessoais. Obrigado por todo amor e carinho no nosso dia-a-dia. Talvez não existam palavras para agradecer tudo o que vocês me proporcionaram, mas podem ter certeza que nada conseguiria sem o apoio e dedicação de vocês.
À Maria Alice, por toda a felicidade, segurança, equilíbrio e paz que tenho ao estar ao seu lado. Obrigado por compartilhar comigo cada minuto que convivemos e que iremos viver. Obrigado pela compreensão e paciência nos inúmeros momentos que fiquei ausente dedicando-me ao trabalho, e quando juntos pelo amor e carinho. Obrigado, por permitir construir a minha vida e meu futuro ao lado de quem eu amo.
RESUMO
O presente estudo investigou a presença de alterações genéticas em pacientes com periodontite agressiva (PA) em uma população da região sudeste do Brasil (1) avaliando a frequência dos polimorfismos rs1537415 no gene GLT6D1, rs61815653 no gene IL10 e rs1333048 no gene ANRIL, localizados em região intronica e reportados previamente em estudos amplos em populações europeias; e (2) avaliando a presença de alterações genéticas por meio do sequenciamento do exoma de famílias (pais-filhos) com histórico de PA. Para o primeiro objetivo a frequência alélica e genotípica dos SNPs rs1537415, rs6667202, e rs1333048 foram avaliados por meio de PCR com sondas TaqMan específicas, em 200 pacientes com PA, 190 com periodontite crônica (PC) e 202 indivíduos saudáveis (SP). Os dados foram analisados pelo teste qui-quadrado e regressão logística. Apenas o SNP rs6667202 (IL10) mostrou associação com PA, sendo o alelo raro (C) detectado em menor frequência nos pacientes com PA quando comparado com os indivíduos saudáveis e PC. Para o segundo objetivo, foi realizado o sequenciamento do exoma em 2 núcleos familiares, cada um com 4 indivíduos, em que um filho (probando) apresentava PA, o outro filho SP, um dos genitores apresentava histórico de PA e o outro genitor ausência de PA. Inicialmente cada família foi avaliada separadamente, e as variantes comuns entre o genitor afetado e o genitor não-afetado foram descartadas, bem como as variantes comuns com o filho não-afetado. Posteriormente, apenas as variantes comuns com o filho afetado foram selecionadas. Em seguida, as variantes comuns entre as duas famílias foram consideradas associadas com a PA. As variantes selecionadas foram validadas por sequenciamento Sanger. Para prospectar a função das variantes selecionadas uma rede de interação proteína-proteína (PPI) foi construída. Avaliações in silico foram realizadas para identificar o impacto de cada variante na estrutura proteica. Após a filtragem 3 SNVs (single nucleotide variations) e 2 indels (inserções/deleções) foram identificados como genes candidatos à PA. Entre os quais, as variações missense rs142548867 [c.668C>T (p.Pro223.Leu)] em EEFSEC, rs574301770 [c.466C>G (p.Arg156Gly)] em ZNF136 e rs72821893 [c.800G>A (p.Arg267His)] em KRT25 foram associadas a PA. Essas variantes foram classificadas como deletérias para a função proteica pelo algoritmo de predição SIFT, enquanto EEFSEC e ZNF136 foram preditas como possivelmente
prejudiciais pelo algoritmo Polyphen. Dois indels com frameshift foram observados nos
pacientes com PA, rs37146475 in GPRC6A [c.2323-2324insT
(p.Tyr775LeufsTer776)], e inserção de GGAGCAG [c.1366_1372insGGAGCAG (p.Ala457fs ou p.Ala457SrfsTer37)] no gene ELN na posição 7:74057389. Na rede de PPI, diversos genes candidatos estavam altamente interconectados entre si, enquanto a análise in silico demonstrou uma alteração estrutural das proteínas EEFSEC e principalmente GPRC6A, na qual a porção intracelular C-terminal da proteína foi perdida.
Pode se concluir que: (1) o SNP rs6667202 (IL10) foi associado a PA na população brasileira, mas não os SNPs rs1537415 (GLT6D1) e rs1333048 (ANRIL); (2) o exoma identificou SNVs do tipo missense rs142548867 (p.Pro223.Leu) em EEFSEC, rs574301770 (p.Arg156Gly) em ZNF136 e rs72821893 (p.Arg267His) em KRT25, e indels rs37146475 (p.Tyr775LeufsTer776) no gene GPRC6A, e p.Ala457fs ou p.Ala457SrfsTer37 no gene ELN como associados a ocorrência da PA.
Palavras-chave: estudos genéticos de associação, doença periodontal,
ABSTRACT
The present study investigated the presence of genetic alterations in patients with aggressive periodontitis (AgP) in a population from the Southeastern region of Brazilian: (1) evaluating the frequency of polymorphisms rs1537415 in the GLT6D1, rs61815653 in IL10 and rs1333048 in ANRIL gene, located in the intronic region and recently reported in large association studies in European populations; (2) evaluating the presence of genetic alterations by exome sequencing of families (parents-children) with a history of AgP. For the first objective, the frequency of alleles and genotypes of rs1537415, rs6667202, and rs1333048 SNPs were assessed by PCR with specific TaqMan probes in 200 patients with AgP, 190 with chronic periodontitis (CP) and 202 healthy individuals (PH). Data were analyzed by the chi-square test and logistic regression. Only the SNP rs6667202 (IL10) showed association with AgP, and the rare allele (C) was detected in a lower frequency in these patients when compared to PH and CP. For the second objective, the exome sequencing was performed in 2 family nuclei, each with 4 individuals, in which one proband had AgP, a SP sibling, one parent with a history of AgP and the other parent without history of AgP. First each family was evaluated separately. The common variants between the affected and the unaffected parent were discarded, as well as the common variants with the unaffected sibling. After this, only the common variants with the affected proband were selected. Finally, only the common variants between the two families were considered associated with AgP. The selected variants were validated by Sanger sequencing. To investigate the function of the selected variants a protein-protein interaction (PPI) network was constructed. In silico evaluations were performed to identify the impact of each variant on the protein structure. After filtering, 3 single nucleotide variations (SNVs) and 2 indels (insertions/deletions) were identified as candidate genes. Among which, three missense variants rs142548867 [c.668C>T (p.Pro223.Leu)] in EEFSEC, rs574301770 [c.466C>G (p.Arg156Gly)] in ZNF136 and rs72821893 [c.800G>A (p.Arg267His)] in KRT25 were associated with AgP. These variants were classified as deleterious to protein function by the SIFT prediction algorithm, while EEFSEC and ZNF136 were predicted to be potentially damaging by the Polyphen algorithm. Two indels with frameshift were observed in patients with AgP, rs37146475 [c.2323-2324insT (p.Tyr775LeufsTer776)] in GPRC6A, and insertion of
GGAGCAG in the ELN gene [c.1366_1372insGGAGCAG (p.Ala457fs or p.Ala457SrfsTer37)]. In the PPI network, several candidate genes were highly interconnected, while in silico analysis demonstrated a structural alteration of the proteins promoted by SNV in EEFSEC and mainly the indel in GPRC6A, in which the portion C-terminal intracellular was deleted.
It can be concluded that: (1) the SNP rs6667202 (IL10), and not the SNPs rs1537415 and rs1333048, was associated with AgP in the Brazilian population; (2) using the family analysis approach, exome sequencing identifies missense SNPs rs142548867 (p.Pro223.Leu) in EEFSEC gene, rs574301770 (p.Arg156Gly) in ZNF136 and rs72821893 (p.Arg267His) in KRT25, as well as indels rs37146475 (p.Tyr775LeufsTer776) in GPRC6A, and p.Ala457fs or p.Ala457SrfsTer37 in ELN gene as associated with AgP.
Key-words: genetic association studies, periodontal disease, genetic
SUMÁRIO
1 INTRODUÇÃO ... 17
2 ARTIGOS ... 21
2.1 Validation of reported GLT6D1 (rs1537415), IL10 (rs6667202), and ANRIL (rs1333048) single nucleotide polymorphisms for aggressive periodontitis in a Brazilian population. ... 21
2.2 Whole-exome sequencing analysis in aggressive periodontitis: a familial screening approach ... 40
3 DISCUSSÃO ... 81
4 CONCLUSÃO ... 85
REFERÊNCIAS ... 86
1 INTRODUÇÃO
A periodontite é uma doença inflamatória complexa de etiologia multifatorial, desencadeada pela resposta imune-inflamatória do hospedeiro aos periodontopatógenos presentes no biofilme subgengival (Armitage 1999; Pihlstrom et al. 2005). Esta patologia é caracterizada pela destruição dos tecidos periodontais de suporte, que inclui osso alveolar, ligamento periodontal e cemento radicular, e que se não tratada pode levar a perda dental (Armitage 1999; Pihlstrom et al. 2005). Existem duas formas principais de periodontite: a periodontite crônica (PC), de prevalência maior, progressão lenta e que apresenta boa resposta à descontaminação mecânica, e a periodontite agressiva (PA), forma particularmente grave de doença periodontal, que acomete indivíduos jovens sistemicamente saudáveis, e é caracterizada por início precoce, rápida progressão, agregação familiar dos casos e pobre resposta às abordagens terapêuticas (Armitage 1999; Deas & Mealey 2010; Albandar 2014). Estudos epidemiológicos apontam que a prevalência da PA pode variar de 0.1% a 6.5% da população mundial (Demmer & Papapanou 2010), sendo que na população brasileira a prevalência é de 5,5% (Susin & Albandar 2005; Susin et al. 2014). Embora a prevalência seja relativamente baixa, esta patologia possui grande impacto na qualidade de vida dos pacientes, uma vez que por apresentar rápida e severa destruição dos tecidos periodontais, frequentemente leva à múltiplas perdas dentais de maneira precoce (Albandar 2014). A deterioração funcional e estética pode levar a alterações comportamentais, como o desenvolvimento de depressão, agravando ainda mais o impacto na qualidade de vida dos indivíduos afetados pela PA (Priyadarshini et al. 2014).
Em pacientes com PA, a exposição aos irritantes locais, biofilme e cálculo subgengival, não explica por si só a acentuada perda óssea alveolar, sugerindo que fatores do hospedeiro estão envolvidos na determinação da susceptibilidade à doença (Meng et al. 2011). Embora fatores microbiológicos, ambientais e comportamentais possam influenciar o risco a PA, a composição genética do indivíduo é um fator crucial no seu desenvolvimento e progressão (Kinane et al. 2005; Meng et al. 2007, 2011). O papel dos fatores genéticos na
patogênese dessa condição é reforçado por sua característica de forte agregação familiar, ou seja, há tendência de se desenvolver casos da doença em uma mesma família (Laine et al. 2012). Estudos com abordagem familiar mostraram que a prevalência de PA entre irmãos pode alcançar no mínimo 40-50% (Marazita et al. 1994). Além disso, estima-se que os fatores genéticos são responsáveis por aproximadamente 50% da variabilidade nos parâmetros clínicos das doenças periodontais, além de estarem associados a presença de microrganismos específicos e maior produção de marcadores inflamatórios (Nibali et al. 2007, 2013; Vieira & Albandar 2014).
Tradicionalmente, as pesquisas focaram na identificação de alterações genéticas como fatores de risco para a periodontite, em genes específicos, selecionados de acordo com o conhecimento prévio sobre o gene candidato (Schaefer et al. 2010; Laine et al. 2012). A despeito desses esforços, os fatores genéticos que contribuem para a patogênese das doenças periodontais, especialmente da PA, ainda não estão totalmente definidos (Laine et al. 2012). Adicionalmente, a evidência atual indica que a PA e a PC são multifatoriais, a exemplo de outras doenças complexas, com o envolvimento de múltiplos genes de pequeno efeito (geralmente mais de 100 genes para doenças complexas) (Laine et al. 2012; Vieira & Albandar 2014).
Por causa dessas limitações, é essencial executar análises amplas e imparciais para identificar novas variantes genéticas associadas a PA (Wilkening et al. 2009; Kitagaki et al. 2016). Isto é realizado pelos estudos de associação ampla do genoma (GWAS – genome wide association study), que são classificados como abrangentes e independentes de hipótese, ou seja, não levam em consideração nenhuma hipótese pré-estabelecida sobre algum mecanismo ou gene envolvidos (Manolio et al. 2008). Tipicamente, novos loci genéticos candidatos são gerados com base nos estudos de GWAS, que necessitam ser validados/replicados futuramente em populações independentes utilizando a abordagem gene-candidato (Loos et al. 2015).
Utilizando essa abordagem, Schaefer et al. (2010) conduziram o primeiro estudo de GWAS em PA, envolvendo indivíduos alemães. Foi observado que o polimorfismo de nucleotídeo único (SNP) rs1537415 localizado no gene GLT6D1 (glycosyltransferase 6 domain containing 1), que controla indiretamente a diferenciação de células Th2, poderia estar relacionada à PA. À
semelhança desse estudo, outras alterações genéticas foram associadas a PA em outros trabalhos. Por exemplo, o polimorfismo rs61815643 no gene da interleucina (IL)-10, e do polimorfismo rs1333048 no gene CDKN2B antisense RNA 1 (ANRIL), o qual possuí função de regulação de mecanismos associados à colonização do biofilme subgengival por periodontopatógenos (Loos et al. 2015).
Recentemente, Kitagaki et al. (2016) conduziram um estudo de sequenciamento de exons do genoma inteiro de pacientes japoneses com PA, e reportaram a associação do SNP rs536714306 no gene GPR126 (G-protein coupled receptor 126) com a PA. O exoma é uma alternativa ao sequenciamento do genoma inteiro, que avalia a porção codificadora de proteínas do genoma. Apesar dessa porção representar apenas 2% do genoma, é onde se encontram 85% de todas as variações consideradas de grande efeito para o fenótipo (Choi et al. 2009; Manolio et al. 2009).
Contudo, nenhum estudo de larga escala genômica foi realizado na população brasileira. Considerando que diferenças étnicas influenciam de maneira significativa a composição genética individual, a realização dessas análises na população brasileira pode resultar na identificação de novas variantes associadas a PA, ajudando a compreender melhor a patogênese dessa condição. Além disso, poderia futuramente permitir identificar indivíduos mais susceptíveis ao desenvolvimento dessa doença e possibilitando a implementação de abordagens preventivas (Laine et al., 2014). Cabe ressaltar que a população brasileira é altamente miscigenada, acarretando na existência de heterogeneidade genética em relação a outras populações, o que reforça a importância de investigar se alterações genéticas relatadas em outras populações também ocorrem na população brasileira (Andia et al. 2013). Além disso, todos os estudos prévios utilizaram a abordagem de caso-controle para identificar as alterações genéticas candidatas associadas a PA. Em outras palavras, até o presente momento, nenhum estudo de associação genética ampla utilizou essa abordagem em famílias, o que poderia ser uma ferramenta extremamente útil baseada na forte agregação familiar e hereditariedade dessa doença.
Diante da falta de consenso das características genéticas associadas à PA, da ausência de estudos de abordagem genética ampla na população
brasileira, da falta de estudos amplos com abordagem familiar e da necessidade de validação de variantes recentemente associadas à PA na população brasileira, o presente estudo avaliou a associação dos polimorfismos rs1537415 no gene GLT6D1, rs61815653 no gene IL10, e rs1333048 no gene ANRIL e investigou a presença de alterações genéticas em pacientes com PA em uma população brasileira por meio do sequenciamento do exoma.
2 ARTIGOS
2.1 Validation of reported GLT6D1 (rs1537415), IL10 (rs6667202), and ANRIL (rs1333048) single nucleotide polymorphisms for aggressive periodontitis in a Brazilian population.
Artigo submetido ao Journal of Clinical Periodontology.
Tiago Taiete1. Marcio Zaffalon Casati1,2. Camila Schimidt Stolf1.
Mônica Grazieli Corrêa2. Mauro Pedrine Santamaria3. Naira Maria Rebelato
Bechara Andere3. Ricardo Della Coletta4. Enilson Antonio Sallum1. Francisco
Humberto Nociti Júnior1. Karina Gonzales Silvério1. Renato Corrêa Viana
Casarin1.
1 Department of Prosthodontics and Periodontics, Periodontics
Division, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil.
2 Department of Periodontics, Paulista University, São Paulo, SP,
Brazil.
3 Department of Diagnosis and Surgery, School of Dentistry, State
University of São Paulo (UNESP), São José dos Campos, Brazil.
4 Department of Oral Diagnosis, School of Dentistry, University of
Campinas, Piracicaba, São Paulo, Brazil
Abstract
Background and objectives: Susceptibility to aggressive periodontitis (AgP) is influenced by genetic factors, although, in Brazilians, no specific single polymorphism nucleotide (SNPs) have been consistently determined. Meanwhile, some susceptibility genes were significantly identified in AgP patients from different European populations, among which rs1537415 (GLT6D1), rs6667202 (IL10), and rs1333048 (ANRIL) are potential SNPs for diagnosis. However, to date, these associations have not been evaluated in the Brazilian population yet. Therefore, the aim of this study was to investigate whether these previously reported associations with AgP could be replicated in a population from Brazil.
Material and methods: The frequency of alleles and genotypes of the
SNPs rs1537415 (GLT6D1), rs6667202 (IL10), and rs1333048 (ANRIL) were analyzed by TaqMan 5’-exonuclease allelic discrimination assays in 200 AgP, 190 chronic periodontitis (CP) and 202 healthy subjects. Differences in genotype and allele frequencies of each SNPs were analyzed using chi-square test and stepwise logistic regression.
Results: SNP rs6667202 (IL-10) showed an association with AgP.
The rare C allele associated to SNP rs6667202 was less frequent detected in AgP patients when compared to healthy and chronic subjects (23.5%, 34.5% and 30.4% - p=0.0394), being the SNP protective to disease occurrence. Moreover, a final logistic model for AgP diagnosis included gender (p=0.003), age (p<0.001) and IL-10 polymorphism (p<0.001) as significant variables. The SNPs rs1537415 in GLT6D1 and rs1333048 in ANRIL did not show association with AgP.
Conclusion: The SNP rs6667202 in interleukin-10 gene, and not the
SNPs rs1537415 in GLT6D1, and rs1333048 in ANRIL, was associated with AgP in the studied Brazilian population. The rare C allele was less frequent detected in AgP patients than in healthy subjects, confirming the protective feature of this SNP in Brazilian cohort.
Keywords: periodontal diseases, genetic variation, genotype, alleles,
genetic markers.
Introduction
Aggressive periodontitis (AgP) is a severe periodontal disease that is characterized by early age of onset, high rate of disease progression, involvement of multiple teeth with a distinctive pattern of periodontal tissue loss and absence of systemic diseases (Albandar 2014; Armitage 1999). In patients with AgP, exposure to local etiological factors usually cannot account for the significant alveolar bone loss, suggesting that host factors are involved in determining susceptibility to the disease (Meng et al. 2011). Although differences in the microbiologic flora or in other environmental factors could have an influence on AgP occurrence, the individual’s genetic background is a crucial factor,
influencing systemic or host response related risk (Kinane et al. 2005; Meng et al. 2011; Vieira and Albandar 2014). Genetic factors regulate the immune system and certain genetic polymorphisms may render the immune system defective and unable to successfully fend off assaults by periodontopathogens (Vieira and Albandar 2014; Yang et al. 2009).
Although genetic studies have focused on identifying single nucleotide polymorphisms (SNPs) as risk factors for AgP, the genetic factors associated with - or contributing to - its pathogenesis have been identified to a limited extent (Laine et al. 2014; Loos et al. 2015). Several studies have been reported in this effort and varying, if not contradictory, results have been shown (Laine et al. 2012; Loos et al. 2015; Zhang et al. 2011). Currently, only a fraction of susceptibility genes is robustly identified, among which GLT6D1, ANRIL and IL-10 (Hashim et al. 2015; Loos et al. 2015; Schaefer et al. 2013; Schaefer et al. 2011; Schaefer et al. 2009; Schaefer et al. 2010), could be cited, all of them enrolled in immune-inflammatory pathways (Teeuw et al. 2015).
The studies that showed statistical evidences of association between AgP and SNPs in GLT6D1 (rs1537415), IL10 (rs6667202), and ANRIL (rs1333048) gene were carried and replicated in different European population (Schaefer et al. 2013; Schaefer et al. 2011; Schaefer et al. 2009; Schaefer et al. 2010). The association between rs1537415 (GLT6D1) and AgP was also observed in a Sudanese population (Hashim et al. 2015). However, these associations were not significant in some populations, also those geographically close. Although IL10 SNP rs6667202 was identified in a German population, and confirmed in a Dutch cohort, this association was not significant in AgP from German-Austrian cohort (Schaefer et al. 2013). Similarly, rs1333048 in ANRIL which was associated with AgP in German and Turkish cohort was not confirmed in an Italian population (Schaefer et al. 2013; Schaefer et al. 2009). Indeed, individuals from different populations carry different profiles of genetic variants and there may be substantial geographic differentiation (Abecasis et al. 2012). Since genotype and allele frequencies can vary between different ethnic populations, a genetic risk factor for disease susceptibility in one population may not be a risk factor in another population (Hashim et al. 2015; Loos et al. 2005;
Meng et al. 2007). In this context, significant SNPs related to some disease should be replicated and validated in independent cohorts.
Therefore, considering the importance of validation of these polymorphisms associations with AgP in other populations, which had a higher prevalence of AgP when compared to the European population (5.5% vs 0.2%); and the great miscegenation of Brazilians, which can significantly influence the individual genetic background, the objective of the present study was to investigate whether previously reported associations between rs1537415 (GLT6D1), rs6667202 (IL10), and rs1333048 (ANRIL - CDKN2B-AS1) with AgP in European populations could be replicated in a population from Brazil.
Material and methods Study Population
The study was designed in accordance with the STROBE Statement for observational studies, and it was approved by the Ethics Committee of the University of Campinas (58679416.4.0000.5418). Informed written consent was granted by each subject after explanations were provided. A total of 592 subjects (200 with AgP, 190 with chronic periodontitis – CP - and 202 healthy subjects) from the Southeastern region of Brazil were recruited from patients referred to the Graduate Clinic of Piracicaba Dental School, University of Campinas, Piracicaba, Brazil, Periodontology Clinic of UNESP, State University of São Paulo, São José dos Campos, Brazil, and Periodontology Clinic of Paulista University, São Paulo, Brazil. All patients received complete intraoral and periodontal examination, including full-mouth plaque index (FMPI), according to Ainamo and Bay (1975), and full-mouth bleeding score (FMBS), according to Mühlemann and Son (1971), probing pocket depth (PPD), clinical attachment level (CAL) at six point around each tooth using a manual probe (PCPUNC 15®, HuFriedy, Chigaco, IL, USA), tooth mobility, radiographic examination, and complete medical and dental questionnaires.
AgP and CP patients were identified on the bases of American Academy of Periodontology criteria (Armitage 1999). Furthermore, these patients
needed to present the presence of at least 20 teeth, at least 8 teeth presenting PD ≥ 5mm with bleeding on probing (having at least 2 with PPD ≥ 7mm), < 35 years of age for AgP patients, and > 35 years for CP. Healthy subjects did not show interproximal attachment loss and having no probing pocket depths of >4mm (Jonsson et al. 2011; Sorensen et al. 2008).
Additional exclusion criteria were as follow: smoking, diabetes, or other systemic diseases (e.g., cardiovascular disease), hepatitis or HIV infection, immunosuppressive chemotherapy, chronic usage of anti-inflammatory drugs, use of orthodontic appliances, and diseases of the oral hard and soft tissues (except caries and periodontitis) (Andia et al. 2013).
Isolation of genomic DNA
The sampling of buccal epithelial cells was performed as described before (Trevilatto and Line 2000). Briefly, the subjects undertook a mouthwash for 60 seconds, containing 5 mL 3% dextrose/glucose solution. The individuals were oriented to rub their tongue on the oral mucosa and teeth. Three mL of TNE solution [17 mM Tris/HCl (pH 8.0), 50 mM NaCl and 7 mM EDTA] diluted in 66% ethanol was added to the samples tube. The samples were centrifugated at 3000 rpm for 10 min, and the supernatant was discarded and the buccal cell pellet resuspended in 500 µL of extraction buffer [10 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.5% SDS]. Genomic DNA was isolated from buccal epithelial cells using a salting-out protocol previously described (Aidar and Line 2007). DNA was dissolved in nuclease-free water, and its quantity was evaluated spectrophotometrically using a Nanodrop 2000 device (Thermo Scientific, Wilmington, DE, USA).
Genotyping
PCR-based genotyping of rs1537415 (GLT6D1), rs6667202 (IL10), and rs1333048 (ANRIL) (table 1) was performed on the LighCycler 480 (Roche Diagnostics GmbH) using TaqMan 5’-exonuclease allelic discrimination assays (Thermo Scientific). PCR were carried out in a total of 10 µl, containing 20 ng of
gDNA, 5 µl TaqMan Genotyping Master Mix, 0.5 µl TaqMan genotyping assay mix 20X. PCRs were performed under the following conditions: 10 min at 95 oC,
40 x (15 sec at 95 oC, 1 min at 60 oC). For quality control purposes, reactions
were randomly repeated in 10% of the samples for each SNP, and the concordance rate was 100% (Machado et al. 2016). All samples were successfully genotyped, with a genotype call rate of 100%.
Statistical Analysis
Demographic and clinical data of patient age, PPD, CAL, FMPI and FMBS were initially evaluated by the Shapiro-Wilk test (for normality). Those presenting a Shapiro-Wilk p-value > 0.05 were analyzed by one-way ANOVA followed by Tukey’s HSD test. Those presenting a Shapiro-Wilk p-value ≤ 0.05 were analyzed by the Kruskal-Wallis/Dunn tests. Comparison among groups with respect to gender was performed by Fisher’s Exact test.
Analysis of Hardy-Weinberg equilibrium was used to test deviation of genotype distribution (BioEstat 5.4, Belém, PA, Brasil). Association analysis between rs1537415 (GLT6D1), rs6667202 (IL-10), and rs1333048 (ANRIL) and AgP was performed using chi-square test. The odds ratio associated with the 95% confidence interval was also calculated. A Stepwise logistic regression was used to determine the best model to AgP diagnosis (dependent variable), considering age, gender, ethnic group (Caucasoid, African American and Asian) and all SNPs (allele and genotype associated to disease) as independent variables. SIGMAstat program was used, considering alpha=5%.
Results
Study Population
Demographic and clinical characteristics of the subjects enrolled in this study is shown in table 2. The mean age of AgP was 34.12 ± 4.6 years, which was statistically younger than that of chronic periodontitis patients 50.0 ± 7.2. However, there was no difference in age between AgP and healthy subjects (30.7 ± 5.8), but healthy subjects were younger than chronic periodontitis subjects
(p<0.05). Statistically significant differences were seen when gender was compared between groups. AgP group presented a greater number of female’s subjects when compared do CP and healthy group. No significant difference was observed between groups regarding ethnic group.
Genotyping
The genotype frequencies observed for all studied polymorphisms in controls did not reveal statistically significant differences compared with those expected under Hardy-Weinberg equilibrium (p>0.05) (table 3).
SNP rs6667202 (IL10) showed an association with AgP in this Brazilian case-control cohort when compared to healthy and CP group. In this group the genotypes frequencies were AA 57.5%, AC 37.8%, and CC 4.7%, resulting in a frequency of 23.5% of the allele C, and carrier frequency of at least one C allele of 42.5%. While in the healthy group the frequency of AA, AC, CC were 41.5%, 48.1% and 10.4% respectively, resulting in a C allele frequency of 34.5%, and carrier frequency of least one C allele of 58.5%. CP showed genotype frequency of AA 50.3%, AC 38.7%, and CC 11.0%, resulting in a frequency of 30.4% of the allele C, and carrier frequency of at least one C allele of 49.7% (Table 3). The C rare allele associated to SNP rs6667202 (IL10) was protective to AgP occurrence, presenting Odds ratio of 0.59 (95%CI 0.42-0.81, p=0.002) and 0.71 (95%CI 0.50-0.98, p=0.05) in relation to healthy and chronic periodontitis respectively. The absence of C allele, i.e., homozygous AA allele, represented increased risk for AgP (OR 1.91, 95%CI 1.25 - 2.92, p=0.004).
Allele and genotype frequencies regarding rs1537415 in GLT6D1 and SNP rs1333048 in ANRIL were not significant for AgP diagnosis in this Brazilian cohort. Interestingly, the SNP rs1333048 in ANRIL represented a risk for CP (OR 1.43, 95%CI 1.05-1.93, p = 0.02).
Stepwise logistic regression construct a model to indicate AgP diagnosis. Considering genotype distribution, AgP diagnosis was associated to age (p<0.001), gender (p<0.005) and SNP rs6667202 (IL10) (p<0.001).
Regarding allele distribution, age (p<0.001), gender (p<0.003) and SNP rs6667202 (IL-10) (p<0.001) (Table 4 and 5).
Discussion
Although genetic factors play a significant role in the pathogenesis of AgP, few genes have been established as AgP risk factors (Loos et al. 2015; Vieira and Albandar 2014). To date, some genetic variants were robustly identified as associated with AgP in European population, among which rs1537415 in GLT6D1, rs6667202 in IL-10, and rs1333048 in ANRIL gene (Laine et al. 2014; Loos et al. 2015; Schaefer et al. 2013; Schaefer et al. 2010). However, the profile of disease modifying genes for AgP may not be equal for different ethnic populations (Laine et al. 2012). In the present study, we investigated whether SNPs in GLT6D1 (rs1537415), IL10 (rs6667202) and ANRIL (rs1333048) could be associated with AgP in a Brazilian population, which present a greater AgP prevalence when compared to European population. The main finding of this study was that genetic variant rs6667202 in the interleukin-10 gene, and not the SNPs in GLT6D1 and ANRIL, was associated with AgP when compared to healthy and CP group in a Brazilian cohort.
The SNP rs6667202 is located upstream region of IL10, and was first associated to AgP in German population, and validated in an independent cohort of Dutch AgP patients (Schaefer et al. 2013). Corroborating Schaefer’s trial (2013), in the present study, the rare C allele was less frequent detected in AgP patients when compared to healthy. Moreover, the present study also compared this SNP’s math to chronic periodontitis population, being this SNP differently distributed between populations. This is an important, and firstly described result, since CP and AgP are phenotypically different diseases and, based on our findings, could present a different genotype background. Moreover, this finding could represent a form to differentiate the risk for these diseases, what, clinically, could help, in future, the development of most individualized treatment and preventive protocols.
To best of our knowledge, this is the first association between AgP and the SNP rs6667202 in IL10 in Brazilian population. A recent study in Brazilians
concluded that the SNPs, rs1800896, rs1800871, and rs1800872 located in upstream region of IL10, previously associated with chronic periodontitis, were not associated with AgP (Silveira et al. 2016). Interestingly, regression analysis drawn a model including IL10 SNP (allele or genotype), along with gender and age, as factors diagnostics to AgP. This result indicate that younger females do not presenting the C allele in IL-10 gene could represent the risk population for AgP. However, some caution should be taken when consider this result. Indeed, age significance is expected due to AgP intrinsic characteristic of youngers-associated disease. Moreover, in our population, there was a higher prevalence of female in AgP cohort. Although several studies have neglected the gender association, this result should be considered in future studies (Laine et al. 2012; Vieira and Albandar 2014).
The IL10 gene encodes the anti-inflammatory cytokine interleukin-10, which plays a role in the regulation of proinflammatory cytokines. IL-10 is widely expressed in inflamed periodontal tissues, in which it is thought to be associated with lower disease severity, once it promotes the suppression of innate inflammatory cytokines (Garlet 2010; Garlet et al. 2006). It can be speculated that the rare C allele, which is enriched in the healthy subjects, results in increased production of IL-10, since previous studies have shown lower IL-10 levels in gingival crevicular fluid of AgP patients (Casarin et al. 2010; Teles et al. 2010). Future studies will be important to fully assess the impact of this SNP on IL-10 production and on the AgP pathogenesis.
Moreover, the results of the present study did not confirm the association of rs1537415 in GLT6D1 and rs1333048 in ANRIL with AgP in the Brazilian population. SNP rs1333048 is in a regulatory region and not a protein-coding region of ANRIL, and it was identified to be associated with generalized and localized AgP in a large cohort of German population (Schaefer et al. 2009), and replicated in a German-Northern Irish population. The GLT6D1 SNP rs1537415 was discovered and replicated in two independent German cohorts, and also replicated in a Sudanese population (Hashim et al. 2015; Schaefer et al. 2010). This SNP is located within intron 2 GLT6D1 gene. Interestingly, the rare allele in the study of Schaefer et al. (2010) was the G allele, which was present in 48.4% in AgP cases and 38.8% in controls, whereas in the present study the
G allele was the common allele present in 64.6% to 65.9% in the three groups evaluated. This difference in the allele frequency between German and Brazilian population emphasizes the role of ethnicity in the individual genetic make-up, which in turn reinforces the importance of replications studies in different populations to understand the genetic basis of AgP.
The lack of association between AgP and rs1537415 (GLT6D1) and rs1333048 (ANRIL) in the present study could be due to some factors. AgP is considered a complex and polygenic disease, therefore, different genetic risk factors can have a similar effect in different populations (Hashim et al. 2015; Loos et al. 2005; Meng et al. 2007). In this vein, the identified SNPs sometimes are genetic markers and not the true causative variants, pointing to a gene locus of importance to disease. This phenomenon could indicate that other polymorphisms, in another region of the same gene, may be associated with the disease in different populations (Loos et al. 2015). Furthermore, the investigation of specific SNPs in candidate genes, as performed in the present study, has some limitations. This approach allows the evaluation of specific genetic information of a particular region of only few genes, while AgP (as CP) is characterized by involvement of various biological pathways and genes. Thus, open-ended and hypothesis-free studies, such as genome-wide association studies (whole genome sequencing or whole exome sequencing), can overcome this limitation by evaluating thousands of genes and genetic loci at the same time (Wilkening et al. 2009).
In conclusion, the SNP rs6667202 in IL10 gene, and not the SNPs rs1537415 in GLT6D1, and rs1333048 in ANRIL, was associated with AgP in the studied Brazilian population. The rare C allele of SNP rs6667202 was less frequent detected in AgP patients than in healthy subjects, confirming that this SNP has protective effect in this Brazilian cohort.
Acknowledgements
The authors would like to thank Isabela Lima França Grohmann, Manuela Rocha dos Santos and Cassia Fernandes Araújo for help us with the patient selection.
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Table 1. Characteristics of the Single-Nucleotide Polymorphisms (SNPs)
SNP Gene Chromosome Position Aleles Original Study
rs1537415 GLT6D1 9 135637876 C/G Schaefer et al. 2010 rs6667202 IL10 1 206783747 A/C Schaefer et al. 2013 rs1333048 ANRIL 9 22125348 A/C Schaefer et al. 2009; 2013
Table 2. Demographic and clinical characteristics.
Characteristics AgP CP Healthy
Age (mean±sd) 34.06 ± 4.6 A 50.0 ± 7.2 B 30.7 ± 5.8 A Gender (% - M/F) 21 / 79 A 34.9 / 65.1 B 35.0 / 65.0 B Ethnic (Af / C / As) 16.5 / 83.5 / 0 A 14.1 / 85.9 / 0 A 13.2 / 85.2 / 1.6 A
FMPI (mean±sd) 23.10±6.53 A 17.05 ± 8.42 A 19.36 ± 5.38 A FMBS (mean±sd) 24.83±9.05 A 23.53 ± 7.69 A 19.39 ± 2.31 A PPD (mean±sd) 2.35±0.05 A 2.28 ± 0.27 A 2.13 ± 0.23 A CAL (mean±sd) 5.44±1.04 A 5.44 ± 0.90 A 4.15 ± 0.72 A
Different letters (capital for intergroup analysis) indicate statistical difference. Kruskal Wallis / Dunn test analyzed age. Fisher’s Exact Test analyzed gender and ethnic group. Comparison among groups with respect to FMPI, FMBS, PPD and CAL was performed by one-way ANOVA / Tukey test.
Sd – Standard Deviation; Gender (M/F) – Gender (Male / Female); Ethnic (C / Af / As) – Ethnic (Caucasoid, African American and Asian); FMPI – Full Mouth Plaque Index; FMBS – Full Mouth Bleeding Index; PPD – Probing Pocket Depth; CAL – Clinical Attachment Level.
Table 3. Allele and genotype frequency of Polymorphisms rs1537415, rs6667202, and rs1333048. SNP HWE (p-value) AgP (%) CP (%) Healthy (%) p value Odds ratio AgPxHealthy (95% CI) p-value Odds ratio AgPxCP (95% CI) p-value
Odds ratio CPxHealthy (95% CI) p-value rs1537415 (GLT6D1) 0.7869 Aleles (C / G) G 65.4 64.6 65.9 0.9739 C 34.6 35.4 34.1 Genotype (CC / CG / GG) CC 11.6 13.0 10.9 0.9864 CG 45.9 44.7 46.3 CC 42.5 42.3 42.8 rs6667202 (IL10) 0.6722 Aleles (A / C) C 23.5 30.4 34.5 0.0394 0.59 (0.42 - 0.81) p=0.002 0.71 (0.50 - 0.98) p = 0.05 n.s. A 76.5 69.9 65.5 Genotype (AA / AC / CC) AA 57.5 50.3 41.5 0.0500 1.91 (1.25 - 2.92) p=0.004 n.s. n.s. AC 37.8 38.7 48.1 n.s. n.s. n.s. CC 4.7 11.0 10.4 0.41 (0.18 - 0.96) P=0.05 0.39 (0.17 - 0.89) P=0.037 n.s. rs1333048 (ANRIL) 0.4131 Aleles (A / C) C 49.8 58.6 50.7 0.035 n.s. 1.43 (1.05 - 1.93) p = 0.02 n.s. A 50.2 41.4 49.3 Genotype (AA / AC / CC) AA 29.4 16.5 24.2 0.128 AC 41.5 49.7 50.0 CC 29.1 34.0 25.8
Table 4. Final Stepwise Regression for the diagnosis of aggressive periodontitis considering genotype.
R RSqr p Age 0.289 0.084 <0.001 Gender 0.346 0.120 0.005 GLT6D1 n.s. IL-10 0.346 0.107 <0.001 ANRIL n.s.
Table 5. Final Stepwise Regression model for the diagnosis of aggressive periodontitis considering allele.
R RSqr p Age 0.289 0.084 <0.001 Gender 0.317 0.101 0.003 GLT6D1 n.s. IL10 0.340 0.116 <0.001 ANRIL n.s.
2.2 Whole-exome sequencing analysis in aggressive periodontitis: a familial screening approach
Artigo a ser submetido na Plos One.
Tiago Taiete1. Marcio Zaffalon Casati1,2. Luciane Martins1. Denise
Carleto Andia1,3. Luciana Souto Mofatto4. Ricardo Della Coletta5. Mabelle de
Freitas Monteiro1. Enilson Antonio Sallum1. Francisco Humberto Nociti Júnior1.
Karina Gonzales Silvério1. Renato Corrêa Viana Casarin1.
1 Department of Prosthodontics and Periodontics, Periodontics
Division, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil.
2 Department of Periodontics, Paulista University, São Paulo, SP,
Brazil.
3 Dental Research Division, School of Dentistry, Paulista University,
São Paulo, SP, Brazil.
4 Department of Genetics, Evolution and Bioagents, Genomic and
Expression Laboratory, Institute of Biology, University of Campinas, Campinas, SP, Brazil.
5 Department of Oral Diagnosis, School of Dentistry, University of
Campinas, Piracicaba, São Paulo, Brazil
Abstract
Aggressive periodontitis (AgP) is a severe form of periodontitis, which presents early onset, rapid progression, and familial aggregation. Although genetic factors are involved in determining susceptibility to AgP, they have been identified to a limited extent. Thus, aim of the present study was to identify genetic variants associated with AgP via whole-exome sequencing (WES) through a familial screening approach. WES was performed in 2 nuclear families, each one with four subjects, including a proband affected by AgP, a healthy sibling, an affected parent, and an unaffected parent. Firstly, each family were analyzed separately. Common variants between the affected parent and the unaffected parent were excluded, as well as common variants from the unaffected sibling. After, only the common variants between this data set and the proband were
selected. Finally, only the common variants between the two families were considered associated with AgP. Candidate genes were further sequenced by Sanger technique to confirm the identified variants sequences. To explore functional molecular correlations among genes with the identified genetic variants, a protein-protein interaction network (PPI) was constructed. Moreover, in silico analysis were done to identify deleterious impact of each variant on protein structure. Three missense single nucleotide variations (SNVs) in exomic region, rs142548867 in EEFSEC [c.668C>T (p.Pro223.Leu)], rs574301770 in ZNF136 [c.466C>G (p.Arg156Gly)], and rs72821893 in KRT25 [c.800G>A (p.Arg267His)], and 2 indels with frameshift, rs37146475 in GPRC6A
[c.2323-2324insT (p.Tyr775LeufsTer776)], and in ELN gene
[c.1366_1372insGGAGCAG (p.Ala457fs or p.Ala457SrfsTer37)] were associated with AgP and presented functional impact in SIFT and Polyphen database. Moreover, PPI analysis showed highly interconnected network, linking SNVs to central nodes, as ubiquitin C. Meanwhile, in silico analysis indicate that a SNV on EEFSEC (p.Pro223.Leu) and GPRC6A (p.Tyr775LeufsTer776) genes, generate altered proteins, especially in GPRC6A where a loss in C-terminal tail were seen. It can be concluded that using a familial filtering approach, missense SNVs in EEFSEC (p.Pro223.Leu), ZNF136 (p.Arg156Gly) and KRT25 (p.Arg267His) genes, and indels in GPRC6A (p.Tyr775LeufsTer776), and ELN ((p.Ala457fs or p.Ala457SrfsTer37) genes were identified and associated to AgP occurrence.
Keywords: genetic association studies, periodontal disease, genetic
variations, genetic markers
Introduction
Aggressive periodontitis (AgP) is characterized by an early age of onset, rapid progression in otherwise healthy individuals, and familial aggregation, which, if left untreated, can lead to early edentulism [1]. Its prevalence ranges from 0.1% to 6.5% in different populations around the world, while in Brazilian population the prevalence reported is 5.5% [2, 3]. Although affecting a relatively small proportion of the population compared with chronic periodontitis (CP) (prevalence of 14% to > 50%), the rapidity of progression and
high risk of tooth loss at a young age make AgP a unique and relevant disease [4-6]. This marked periodontal destruction cannot be explained only by the exposure to local irritants (i.e., subgingival biofilm and calculus), suggesting that host factors are involved in determining susceptibility to AgP [7].
There is evidence that individual’s genetic make-up is a crucial factor influencing host susceptibility to AgP [7-10], which is highlighted by the familial aggregation, that is one of the primary features of AgP [11]. It is noteworthy that the prevalence of AgP is disproportionately high among certain families, with some studies reporting that the percentage of affected siblings may reach 40-50% or even higher [7]. However, although many studies have been carried out to investigate the association between genetic factors and AgP, varying and often contradictory results were found [11-13]. This recognition of genetic factors those contribute to disease susceptibility in AgP is important to understand its pathogenesis, and could help develop new diagnostics tools and therapy strategies for prevention and treatment of the disease [10, 11, 13, 14].
Currently, specific genetic factors associated with to AgP have been identified to a limited extent [11, 12]. Previous studies evaluated specific single nucleotide polymorphisms (SNPs) in only a few genes, and have not captured the complete genetic information of a particular region of interest [11, 12]. Because of these limitations, it is essential to carry out unbiased large scale analysis to identify novel disease-associated genetic variants for AgP [14, 15]. The emergence of genome-wide association studies (GWAS) using whole genome sequencing (WGS) or whole exome sequencing (WES) is improving comprehensive, open-ended and hypothesis-free studies. However, regarding AgP disease, scarce information was provided [11, 14].
Schaefer et al. [16] conducted a GWAS in a German population and reported the association between the intronic SNP rs1537415 of the GLT6D1 gene with the AgP. Recently, Kitagaki et al. [14] conducted a WES in a Japanese population with AgP, and found that this disease could be associated with SNP rs536714306 of GPR126. However, no GWAS was performed in Brazilian population. In addition, one of most important characterisc of AgP, the familial aggregation, have never been considered in data analysis. Since that AgP is thought to be a hereditary trait and show a clear familial aggregation, the
family-based approach became extremely valuable. Thus, in view of these points, the present study aimed to investigate AgP genetic variations studying familial nuclei via whole-exome sequencing.
Material and Methods Subjects
The study was designed in accordance with the STROBE Statement for observational studies, and it was approved by the Ethics Committee of the University of Campinas (58679416.4.0000.5418). Subjects from 2 nuclear families, each one with four subjects, from the Southeastern region of Brazil were recruited from the patients referred to the Graduate Clinic of Piracicaba Dental School, University of Campinas, Brazil, between September 2014 and November 2014. Informed written consent was granted by each subject after explanations were provided.
All patients received complete intraoral and periodontal examination, including full-mouth plaque index (FMPI), according to Ainamo and Bay [17], and full-mouth bleeding score (FMBS), according to Mühlemann and Son [18], tooth mobility, radiographic examination, and complete medical and dental questionnaires. Additionally, for the parents, periodontal records were recovered from dental records. The diagnosis of AgP and CP was based on the American Academy of Periodontology criteria [19]. The study was accomplished with two probands affected by AgP, their parents (one with a history of AgP, one without a history of AgP) and their sibling (healthy subject – without AgP), as described below and in Fig1:
AgP Probands – Subjects presented periodontal pockets with clinical
attachment loss (CAL) and radiographic bone loss in at least three teeth different from 1st molars and incisors, at least eight teeth with a probing pocket depth
(PPD) ≥ 5 mm and bleeding on probing. At least two of the eight qualifying teeth must have PPD ≥ 7 mm; age inferior to 35 years.
Non-AgP siblings – Healthy subjects, who did not show any sites with
CAL and PPD ≥ 3 mm and presented FMBS < 25%; tooth mobility was not found; age inferior to 35 years.
AgP affected parents – the progenitors who presented the same
periodontal characteristics as AgP probands, but had experienced multiple tooth loss due to disease progress; also, they were older than 35 years. The diagnosis was obtained after careful intra-oral examination and evaluation of past radiographic and dental records. Periodontal characteristics of AgP parents were recovered from their dental records.
Non-AgP parents – the progenitors who did not presented any
characteristic of AgP, but they had slight or moderate CP.
Additionally, the exclusion criteria were: smoking, systemic diseases (e.g., diabetes, hepatitis or HIV infection), diseases of the oral hard or soft tissues (except caries and periodontitis), use of orthodontic appliances and immunosuppressive chemotherapy [20].
Isolation of genomic DNA
The sampling of buccal epithelial cells was performed as described by Trevilatto & Line [21]. DNA was then purified by sequential phenol/chloroform extraction and salt/ethanol precipitation, dissolved in nuclease-free water, and its concentration was evaluated spectrophotometrically using a Nanodrop 2000 device (Thermo Scientific, Wilmington, DE, USA). DNA integrity was evaluated using 1% agarose gel electrophoresis.
Exome Analysis
Whole-exome sequencing (WES) was performed using the Illumina’s next-generation sequencing technology. Library preparation was performed according to the Nextera Rapid-Capture Exome kit (Illumina, San Diego, CA, USA), and the sequencing was performed in Illumina HiSeq 2500 system (Illumina Inc). The samples were sequenced using the Illumina HiqSeq 2500
