Paula Rocha Moreira

Methylation of P16, P21, P27, RB1 and P53 genes in odontogenic keratocysts

BACKGROUND Odontogenic keratocyst (OKC) is a benign neoplasm with an aggressive clinical behavior and a high recurrence rate. Although epigenetic alterations have been reported in different tumors, these events were not investigated in OKC yet. The aim of this study was to investigate the presence of methylation in P16, P21, P27, P53 and RB1 genes in OKC tumors. METHODS Methylation-specific polymerase chain reaction (MSP) was used to evaluate the presence of methylation in 10 samples of OKCs, 10 samples of dental follicles and six samples of normal mucosa. RESULTS The methylation status of the P16 gene was similar among the three groups. In P21 gene, 30% of OKCs were methylated while no methylation could be detected in the other groups. High frequency of P27 methylation (90%) was observed in dental follicles, however, some OKC lesions (10%) and normal mucosa samples (33%) were also methylated. Concerning the RB1 gene, positive results were detected only in dental follicles (40%). No positive result was observed considering P53 gene. CONCLUSIONS Our data show methylation of the promoter of P21 gene in OKCs. In addition, methylation of the P27 and RB1 genes are commonly found in dental follicles. Further studies are necessary to determine the functional relevance of these alterations.

Interleukin-10 Gene Polymorphism (-1082G/A) is Associated with Toxoplasmic Retinochoroiditis

PURPOSE Experimental data have demonstrated a relevant role for IL-10, an anti-inflammatory cytokine, in the modulation of acute ocular toxoplasmosis. Therefore, this study was conducted to investigate the possible association between an IL10 gene polymorphism at position -1082 and toxoplasmic retinochoroiditis (TR) in humans. METHODS One hundred patients with diagnosed TR were recruited from the Uveitis Section, Federal University of Minas Gerais. For comparison, one hundred healthy blood donors with positive serology for toxoplasmosis and without retinal signs of previous TR were included in the study. Genomic DNA was obtained from oral swabs of individuals and amplified using polymerase chain reaction (PCR) with specific primers flanking the locus -1082 of IL10 (-1082G/A). PCR products were subjected to restriction endonuclease digestion and analyzed by polyacrylamide gel electrophoresis, to distinguish allele G and A of the IL-10 gene, allowing the detection of the polymorphism and determination of genotypes. RESULTS There was a significant difference in the genotype distribution between TR patients and control subjects (chi(2) = 6.33, P = 0.04). Carriers of the IL10 -1082 A allele (AA+AG genotypes) were more often patients with TR than control subjects (chi(2) = 5.97, P = 0.01, OR, 2.55; 95% CI, 1.11 < OR < 5.55). In a subgroup analysis, there was no significant difference in genotypes and allele carriage regarding visual acuity, involvement of both eyes and TR recurrence. CONCLUSIONS This study suggests that the genotypes related with a low production of IL-10 may be associated with the occurrence of TR.

Epigenetics and periodontal disease: future perspectives

Periodontitis is a multifactorial infection characterized by inflammation and destruction of tooth supporting tissues, as a result of the response of a susceptible host to bacterial challenge. Studies have demonstrated that epigenetic events are able to influence the production of cytokines, contributing to the development of inflammatory diseases. Epigenetic events act through the remodeling of chromatin and can selectively activate or inactivate genes, determining their expression. The epigenetic process, by inducing a change in cytokine profile, may subsequently influence the pathogenesis and determine the outcome of many infectious diseases. These findings may have relevance for inflammatory diseases in which the expression of cytokines is unregulated. The purpose of this review is to show evidence that supports the hypothesis that epigenetic alterations, such as hyper and hypomethylation, of cytokine genes, could help to understand the mechanisms related to periodontal disease activity. Therefore, epigenetics may have future impact on diagnosis and/or therapeutics of periodontal disease.

Expression, polymorphism and methylation pattern of interleukin-6 in periodontal tissues

Periodontitis is considered an inflammatory disorder of bacterial etiology that results in periodontal tissue destruction, as a result of complex interactions between periodontal pathogens, host and immune response. Genetic and epigenetic mechanisms may modulate the individual response since it is able to influence the gene expression. The aim of this study was to evaluate the impact of -174 G/C polymorphism and the methylation status of the promoter region of IL-6 gene on the expression of IL-6 in gingival samples from individuals with chronic periodontitis. Gingival biopsies were collected from 21 patients with chronic periodontitis and 21 controls. Histologic sections stained by hematoxylin-eosin were used for histopathological evaluation. The IL-6 gene expression was assessed by quantitative real-time PCR. The polymorphism IL-6 -174 C/G was studied by polymerase chain reaction (PCR) amplification and restriction endonuclease digestion (HspII). Methylation-specific polymerase chain reaction was used to verify the DNA methylation pattern. The number of inflammatory cells in tissue fragments from individuals with chronic periodontitis was higher than in the control group and the inflammatory infiltrate was predominantly mononuclear. The expression of IL-6 was higher in the group with periodontitis. In polymorphism assay, no statistical difference in the distribution of genotypes and alleles in both groups were observed. The most of samples were partially methylated. No difference was observed in methylation pattern from two different regions of the IL-6 gene among groups. The high expression of IL-6 is an important factor related to chronic periodontitis, but was not associated with methylation status or the -174 (G/C) genetic polymorphism, suggesting that other mechanisms are involved in this gene transcription regulation.

Evaluation of IL17A expression and of IL17A, IL17F and IL23R gene polymorphisms in Brazilian individuals with periodontitis

The IL23/Th17 axis plays an important role in the pathogenesis of cell-mediated tissue damage caused either by autoimmunity or immune responses against bacterial infection. Single nucleotide polymorphisms in the IL17A, IL17F and IL23R genes have been associated with several inflammatory diseases. However, these polymorphisms have not yet been studied in periodontitis. The aim of present study was to evaluate the expression of IL17A and occurrence of the IL17A (rs2275913), IL17F (rs763780) and IL23R (rs11209026) gene polymorphisms in different clinical forms or severity of periodontitis in a sample of Brazilian individuals. Peripheral blood was obtained from 30 non-smoker individuals and analyzed by flow cytometry to determine IL-17 expression. Genomic DNA was obtained from oral swabs in 180 individuals and analyzed by Real-time PCR. The study group was composed by individuals without periodontitis (control), with aggressive periodontitis (AP) and with chronic periodontitis (CP). Higher frequency of IL17A+CD4+ T cells was observed in control group. The A+ genotype from IL17A (rs2275913) was associated with lack of disease. No association was found considering the IL17F and IL23R polymorphisms. Our data suggest that IL17A and the presence of IL17A (rs2275913) A allele are associated with the absence of periodontal disease.

Methylation frequencies of cell-cycle associated genes in epithelial odontogenic tumours

OBJECTIVE The benign epithelial odontogenic tumours constitute a group of lesions derived from epithelial elements of the tooth-forming apparatus. This group includes lesions of different biological behaviour, such as ameloblastoma, calcifying cystic odontogenic tumour (CCOT) and adenomatoid odontogenic tumour (AOT). The pathogenesis of these neoplasms remains uncertain and the occurrence of methylation in cell-cycle related genes may be involved in their development. The aim of this study was to investigate the methylation status of P16, P21, P27, P53 and RB1 genes in epithelial odontogenic tumours. DESIGN Methylation-specific polymerase chain reaction (MSP) was used to evaluate the presence of methylation in 13 samples of ameloblastoma, six samples of CCOT, three samples of AOT and 14 samples of dental follicles, included as control. RESULTS Our results showed a distinct methylation profile in each group. In ameloblastoma, the highest methylated genes were P16 and P21, while in CCOT the P21 and RB1 genes were the most commonly methylated genes. Only the P16 and P21 genes were methylated in the AOT samples. In the dental follicle samples, P16, P27 and RB1 genes were commonly methylated. A high percentage of the odontogenic tumours analysed showed methylation of the P21 gene, in contrast to dental follicles. CONCLUSIONS Epithelial odontogenic tumours show a distinct methylation profile in cell-cycle associated genes. In addition to this, the current findings show that epigenetic alterations are common events in epithelial odontogenic tumours.

Methylation Pattern of the IFN-γ Gene in Human Dental Pulp

INTRODUCTION DNA methylation is characterized by the addition of methyl groups in cytosines within cytosine-phosphate-guanine (CpG) islands. Unmethylated islands are related with transcriptionally active structure, whereas methylated DNA recruits methyl-binding proteins that promotes chromatin compaction. Although epigenetic events can influence the expression of cytokines, such events have not been investigated in dental pulp yet. The purpose of the present study was to evaluate the methylation status of the interferon gamma (IFN-gamma) gene in human dental pulp affected by inflammation compared with pulp tissue of impacted molar teeth and to verify the impact of methylation status in the expression pattern of the gene. METHODS Methylation-specific polymerase chain reaction (MSP) was used to verify the DNA methylation status of the IFN-gamma gene in 16 human dental pulps affected by inflammation and in 16 pulp samples of impacted molar teeth. Histologic sections stained by hematoxylin-eosin were used for histopathological evaluation, and the expression of IFN-gamma was assessed by quantitative real-time PCR (qPCR). RESULTS Although total methylation was observed in 43.75% of the samples of normal dental pulp tissues, partial methylation or unmethylation was found in 93.75% of the samples of inflamed pulp tissues. All the samples with total methylation in MSP showed no transcription of IFN-gamma. The qPCR results showed expression of IFN-gamma in 5 of 10 samples with partial methylation. CONCLUSION The present study gives the first evidence of the possible participation of epigenetic events in the development of dental pulp inflammation.

PTCH1 isoforms in odontogenic keratocysts

Odontogenic keratocyst (OKC) is an aggressive benign odontogenic neoplasm associated with PTCH1 alteration. PTCH1 has several isoforms generated by use of different first exon (1b, 1d and 1e). These isoforms code for proteins with different functions, expression profiles and transcriptional regulation. The aim of the present study was to investigate the expression of PTCH1 first exons in OKC tumors to shed light on scenery whereby PTCH1 coordinates OKC tumorigenesis. Forty OKC, including 12 sporadic and 28 associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS), were included in the study. The variants 1b, 1d and 1e were investigated by RT-PCR. The exon 1b was detected in 90% of OKC and none of the dental follicle (control). Most of the OKC, sporadic and syndromic, and all of the samples of dental follicles demonstrated the expression of 1d mRNA. All primary OKC had 1b mRNA while 4 (24%) lesions marsupialized lost 1b expression. In addition, the pattern of exon 1 expression observed in oral mucosa adjacent to the OKC was similar to the OKC tumor. In conclusion, this report showed overactivity of Hedgehog (HH) pathway in OKC lesion and at adjacent oral mucosa. We also demonstrated that marsupialization could alter PTCH1 variants profiling in some OKC cases.

Methylation pattern of IFN-γ and IL-10 genes in periodontal tissues.

UNLABELLED DNA methylation is characterized by the addition of methyl groups in cytosines within CpG islands. Unmethylated CpGs are related to transcriptionally active structure, whereas methylated CpG recruits methyl-binding proteins that promote chromatin compaction. DNA methylation can influence the expression of cytokines and affect the development of periodontal disease. OBJECTIVES The purpose of the present study was to evaluate the methylation status of the interferon gamma (IFN-γ) and interleukin-10 (IL-10) genes in periodontal tissues. DESIGN Methylation-specific polymerase chain reaction (MSP) and DNA sequencing analysis were used to verify the DNA methylation status of the IFN-γ and IL-10 genes, respectively, in samples from subjects without periodontitis and individuals with chronic periodontitis. Histological sections stained by hematoxylin-eosin were used for histopathological evaluation of samples. RESULTS The methylation status of the IFN-γ and IL-10 genes was similar among the groups. Most of the samples were positive for IFN-γ methylation. Only 11% of the periodontitis group showed unmethylated DNA. Considering the IL-10 gene, no unmethylated sample was observed. The profile of total or partial methylation was detected in CpGs evaluated. CONCLUSIONS The results showed evidence that methylation of IFN-γ and IL-10 genes is a usual feature on periodontal tissues. Further studies are needed to determine the functional relevance of these alterations.

Implications of cytokine gene polymorphisms on the orchestration of the immune response: Lessons learned from oral diseases

Over the past 10 years, a plethora of information concerning the influence of gene polymorphisms on cytokine expression has been made available in the literature. Significant contribution to this field has come from studies of oral diseases, one of the widest spread health problems in the world, affecting hundreds of millions worldwide. Here we will discuss the importance of studies of gene polymorphism towards the identification of susceptible groups or prognostic indicators of oral disease. Additionally, we will highlight the differences in data obtained from genetically diverse populations and review the application of cytokine gene polymorphisms studies in oral diseases in autoimmune processes and parasitic infections.