Tecnologias moleculares no estudo das doenças periodontais

Orientador: Márcio Zaffalon CasatiDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A periodontite agressiva é caraterizada por apresentar início precoce, rápida progressão e pobre resposta as abordagens terapêuticas quando comparado a periodontite crônica. Entretanto, os mecanismos responsáveis por essas diferenças ainda não são completamente compreendidos. Ferramentas como a genômica, proteômica ou transcriptômica podem esclarecer esses aspectos, gerando importantes informações a respeito da patogênese das doenças periodontais. Portanto, os objetivos do presente estudo foram: i) Apresentar uma revisão de literatura focada na aplicação da genômica, transcriptômica, proteômica e metabolômica no estudo das doenças periodontais. ii) Avaliar as diferenças no perfil de expressão gênica da mucosa mastigatória, sem a influência do biofilme subgengival, em indivíduos com histórico de periodontite agressiva e crônica, comparando-os entre si e também com o perfil de expressão de indivíduos sem histórico de periodontite visando à identificação de possíveis alterações constitutivas na expressão de genes que podem estar relacionadas com as diferenças entre as duas formas de periodontite. Para o primeiro objetivo, foi realizada uma revisão dos artigos que empregaram as tecnologias ômicas no estudo das doenças periodontais. Os estudos presentes na literatura indicaram que essas tecnologias podem levar a um melhor entendimento dos eventos moleculares envolvidos na patogênese das doenças periodontais. Para o segundo objetivo, foram obtidas amostras teciduais da mucosa mastigatória de 4 pacientes com histórico de periodontite crônica, 4 com periodontite agressiva generalizada e 4 indivíduos sem histórico de periodontite. As amostras foram manipuladas para a extração de RNA total, síntese de cDNA de fita dupla e a hibridização por microarranjo de DNA, avaliando a expressão de 45033 genes. Os programas R e Bioconductor foram empregados para realizar a análise de RMA (Robust-Multi-Array), calcular o valor de expressão (fold-change) e o valor de p para comparações múltiplas através do método de Benjamini-Hochberg. O enriquecimento dos dados, através de ontologia gênica e análise de via, foi realizado com o programa de bioinformática DAVID (Database for Annotation, Visualization and Integrated Discovery). Três comparações foram realizadas: comparação 1 (periodontite agressiva x indivíduos sem histórico de periodontite); comparação 2 (periodontite crônica x indivíduos sem histórico de periodontite); e comparação 3 (periodontite crônica x periodontite agressiva). A comparação entre os grupos identificou: 192 genes e 50 grupos diferencialmente expressos na comparação 1, 43 genes e 27 grupos na comparação 2 e 168 genes e 75 grupos na comparação 3. Genes com funções no sistema imune, entre os quais receptores de linfócitos natural killer, foram mais expressos em periodontite agressiva; em contraste, genes envolvidos na proliferação e diferenciação de queratinócitos, assim como genes com função em processos neurais foram menos expressos. Já os indivíduos com periodontite crônica se caracterizaram por um aumento na expressão de genes com função na resposta a estímulos externos, e uma diminuição na expressão dos genes relacionados ao sistema imune. Dentro dos limites desse estudo, pode-se concluir que existem diferenças nos perfis de expressão gênica da mucosa mastigatória de indivíduos com histórico de periodontite agressiva e crônica, quando comparadas entre si e com indivíduos sem histórico de periodontite, que podem estar associadas às diferenças em suas patogênesesAbstract: Aggressive periodontitis is characterized by early onset, rapid progression and poor response to treatment compared to chronic periodontitis. However, mechanisms responsible for these differences are not fully understood. Tools such as genomics, proteomics and transcriptomics can clarify these aspects, producing important information about the pathogenesis of periodontal diseases. Therefore, the aims of this study were: i) Provide a literature review focused on the application of genomics, transcriptomics, proteomics, and metabolomics in the study of periodontal diseases. ii) Evaluate the gene expression profile of tissue from masticatory mucosa, without the influence of biofilm, in patients with a history of generalized aggressive and chronic periodontitis, and also with gene expression profile of individuals without periodontitis, with the aim to identify possible constitutive alterations in gene expression, which may be related to the differences between both forms of periodontitis. For the first objective, it was performed a review of articles that employed omics technologies in the study of periodontal diseases. The studies in the literature indicated that these technologies can lead to a better understanding of molecular events involved in the pathogenesis of periodontal diseases. For the second objective, masticatory mucosa tissue samples were obtained from 4 patients with a history of chronic periodontitis, 4 with generalized aggressive periodontitis, and 4 from subjects without history of periodontitis. Tissue samples were processed for total RNA extraction, double-strand cDNA synthesis for subsequent hybridization reaction on microarrays, which assessed the expression of 45033 genes. R and Bioconductor softwares used to perform the RMA analysis (Robust Multi-Array), calculate the value of expression (fold-change) and the p-value for multiple comparisons through Benjamini-Hochberg method. Enrichment analysis, through gene ontology (GO) and pathway analysis, was performed with DAVID (Database for Annotation, Visualization and Integrated Discovery). Three comparisons were performed: comparison 1 (aggressive periodontitis x healthy subjects); comparison 2 (chronic periodontitis x healthy subjects); and comparison 3 (chronic periodontitis x aggressive periodontitis). Comparisons analysis found 192 genes and 50 groups differentially expressed in comparison 1, 43 genes and 27 groups in comparison 2, and 168 genes and 75 groups in comparison 3. Genes with function in the immune system, including natural killer cells receptors, were higher expressed in aggressive periodontitis; in contrast, genes involved in proliferation and differentiation of keratinocytes, as well as genes with function in neural process were lower expressed. Chronic periodontitis subjects were characterized by increased expression of genes related to response to external stimuli, and a decrease in the expression of genes related to the immune system. Within the limits of this study, it can be concluded that there are differences in the gene expression profile of masticatory mucosa of patients with a history of chonic and aggressive periodontitis, when compared among them and with healthy group, which may be associated with differences in their pathogenesisMestradoPeriodontiaMestre em Clínica Odontológic

Association of rs142548867 (EEFSEC) and periodontitis Grade C in a young Brazilian population

Periodontitis Stage III-IV, Grade C (PerioC) is a severe form of Periodontitis. The individual genetic background has been shown to be an important etiopathogenic factor for the development of this disease in young, systemically healthy, and non-smokers patients. Recently, after exome sequencing of families with a history of the disease, PerioC was associated with three single nucleotide variations (SNVs) – rs142548867 (EEFSEC), rs574301770 (ZNF136), and rs72821893 (KRT25) – which were classified as deleterious or possibly harmful by prediction algorithms. Objective: Seeking to validate these findings in a cohort evaluation, this study aims to characterize the allele and genotypic frequency of the SNVs rs142548867, rs574301770, and rs72821893 in the Brazilian population with PerioC and who were periodontally healthy (PH). Methodology: Thus, epithelial oral cells from 200 PerioC and 196 PH patients were harvested at three distinct centers at the Brazilian Southern region, their DNA were extracted, and the SNVs rs142548867, rs574301770, rs72821893 were genotyped using 5′-nuclease allelic discrimination assay. Differences in allele and genotype frequencies were analyzed using Fisher’s Exact Test. Only the SNV rs142548867 (C > T) was associated with PerioC. Results: The CT genotype was detected more frequently in patients with PerioC when compared with PH subjects (6% and 0.5% respectively), being significantly associated with PerioC (odds ratio 11.76, p=0.02). Conclusion: rs142548867 represents a potential risk for the occurrence of this disease in the Brazilian population

Association of rs142548867 (EEFSEC) and periodontitis Grade C in a young Brazilian population

Abstract Periodontitis Stage III-IV, Grade C (PerioC) is a severe form of Periodontitis. The individual genetic background has been shown to be an important etiopathogenic factor for the development of this disease in young, systemically healthy, and non-smokers patients. Recently, after exome sequencing of families with a history of the disease, PerioC was associated with three single nucleotide variations (SNVs) – rs142548867 (EEFSEC), rs574301770 (ZNF136), and rs72821893 (KRT25) – which were classified as deleterious or possibly harmful by prediction algorithms. Objective Seeking to validate these findings in a cohort evaluation, this study aims to characterize the allele and genotypic frequency of the SNVs rs142548867, rs574301770, and rs72821893 in the Brazilian population with PerioC and who were periodontally healthy (PH). Methodology Thus, epithelial oral cells from 200 PerioC and 196 PH patients were harvested at three distinct centers at the Brazilian Southern region, their DNA were extracted, and the SNVs rs142548867, rs574301770, rs72821893 were genotyped using 5′-nuclease allelic discrimination assay. Differences in allele and genotype frequencies were analyzed using Fisher’s Exact Test. Only the SNV rs142548867 (C > T) was associated with PerioC. Results The CT genotype was detected more frequently in patients with PerioC when compared with PH subjects (6% and 0.5% respectively), being significantly associated with PerioC (odds ratio 11.76, p=0.02). Conclusion rs142548867 represents a potential risk for the occurrence of this disease in the Brazilian population

Análise da influência do estresse ácido, nutricional e populacional na síntese e atividade de substâncias semelhantes às mutacinas produzidas por isolados clínicos de Streptococcus mutans

A maioria dos isolados clínicos de S. mutans é capaz de sintetizar substâncias antimicrobianas denominadas mutacinas. Este fator de virulência pode ser essencial na colonização e na prevalência da espécie mais relacionada com o desenvolvimento da carie dental, principalmente em nichos de alta complexidade ambiental e competitividade microbiana, como o biofilme dental. Esta pesquisa teve como objetivo principal, analisar a influencia de condi96es ambientais como estresse ácido, nutricional e populacional, na produção de substancia semelhantes as mutacinas in vitro. Os resultados esclareceram os mecanismos fisiológicos de regulação da síntese e/ou atividade de substancias inibitórias semelhantes as mutacinas, em condições ambientais, comumente descritas em um biofilme cariogênico

Abordagens moleculares para diagnóstico em periodontia : o papel do conhecimento ômico

As doenças periodontais são altamente complexas e possuem etiologia multifatorial, com a participação de inúmeros mecanismos biológicos. Esta revisão tem por objetivo discutir alguns dos avanços nas pesquisas científicas que utilizam métodos de biologia molecular para avaliar os aspectos da genômica, proteômica, transcriptômica e metabolômica da doença periodontal. Diversos estudos na literatura demonstram que essas ferramentas podem promover uma melhor compreensão dos mecanismos envolvidos na patogênese e na identificação de novos biomarcadores das doenças periodontais, que podem ser utilizados testes diagnósticos futuros para detectar a presença de doença ativa, predizer progressão futura e avaliar a resposta a terapia periodontal.Periodontal diseases are highly complex and have multifactorial etiology processes with the participation of several biological mechanisms. This review sought to discuss some of the advances in scientific research that use molecular biology methods to address the aspects of genomic, proteomic, transcriptomic, and metabolomic of periodontal diseases. Several studies in the literature show that these tools aim to promote a better understanding of the mechanisms involved in the pathogenesis and identification of new biomarkers of periodontal diseases that may be used in future diagnostic tests to detect the presence of active disease, predict future progression, and evaluate the response to periodontal therapy

Evaluation of genetic alterations in patients with aggressive periodontitis in the brazilian population

Orientador: Renato Corrêa Viana CasarinTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: 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 PAAbstract: 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 AgPDoutoradoPeriodontiaDoutor em Clínica Odontológica140967/203-3CNP

Amoxicillin/metronidazole Associated With Nonsurgical Therapy Did Not Promote Additional Benefits In Immunologic Parameters In Generalized Aggressive Periodontitis: A Randomized Controlled Clinical Trial

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)This randomized, blinded, placebo-controlled clinical trial compared the levels of interferon. (IFN-gamma), prostaglandin E-2 (PGE 2), and interleukin 6 (IL-6) in the gingival crevicular fluid (GCF) from generalized aggressive periodontitis (GAgP) patients treated with nonsurgical therapy associated or not with amoxicillin/metronidazole adjunctive. Method and Materials: Thirty-nine GAgP patients were followed during 6 months. The patients were randomly allocated to one of the groups: experimental (scaling and root planing plus 375 mg amoxicillin and 250 mg metronidazole for 7 days) and control (scaling and root planing + placebo). Probing pocket depth (PPD), relative clinical attachment level (rCAL), gingival margin position (GMP), and IL-6, IFN-gamma, and PGE 2 levels in GCF were evaluated at baseline, and at 3 and 6 months after treatment. Results: Both therapies promoted PPD reductions, rCAL gains, and recession in GMP at the end of the study, with the experimental group presenting an additional PPD reduction in full-mouth analysis and deep pockets at the 3- and 6-month follow-ups (P < .05). During the period of the study, only the experimental group promoted a reduction in PGE 2 levels in deep pockets at 3 and 6 months, while IFN-gamma and IL-6 levels remained unchanged. However, the differences in the immunologic parameters were not statistically significant among the groups. Conclusion: It can be concluded that amoxicillin/metronidazole associated with nonsurgical therapy promotes an additional PPD reduction in the treatment of GAgP; however, this therapy did not promote additional benefits in the evaluated immunologic parameters.474281292Foundation for Research Support of Sao Paulo (FAPESP) [06/60314-5]National Council for Scientific and Technological Development (CNPq) [30878/2006-2]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Original research Periodontics, Microbiology Fabiano Ribeiro CIRANO (a) Effect of Resveratrol on periodontal pathogens during experimental periodontitis in rats

Abstract: The objective of this study was to investigate the antibacterial effect of resveratrol against putative periodontal pathogens during the progression of experimental periodontitis in rats. Periodontitis was induced in rats in one of the first molars chosen to receive a ligature. Animals were assigned to one of two groups: daily administration of the placebo solution (control group, n = 12) or 10 mg/Kg of resveratrol (RESV group, n = 12). The therapies were administered systemically for 30 days, for 19 days before periodontitis induction and then for another 11 days. Then, the presence and concentrations of Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans in the cotton ligatures collected from the first molars were evaluated using real-time PCR. Inter-group comparisons of the microbiological outcomes revealed that no differences were detected for P. gingivalis, T. forsythia and A. actinomycetemcomitans levels (p &gt; 0.05). Continuous use of resveratrol did not promote additional benefits in microbiological outcomes during experimental periodontitis in rats