Expression of chronic pain genes in apical periodontitis tissues.

AIM: To evaluate the expression of genes involved in chronic pain conditions in apical periodontitis (AP) tissues. METHODOLOGY: An electronic search was performed in Scopus and MEDLINE (via PubMed) databases to identify articles (n = 173) related to genes involved in chronic pain conditions. Full-text reviews of the selected articles allowed the prioritization of 16 genes to be investigated with regards to their expression in AP tissues. Periapical lesions (n = 42) were collected during surgical endodontic procedures and processed for mRNA extraction and investigation of target genes via RT-qPCR. Healthy periodontal ligament tissues obtained from third molar extractions were used as controls. Relative levels of target gene expression in AP and control tissues were normalized to GAPDH expression and compared using the 2-ΔΔCt method. Data analysis was performed using the Mann-Whitney U test with a statistical significance threshold set at p < .05. RESULTS: mRNA expression levels of MMP9, TIMP1, TNFA, CAMK4 and COMT were significantly increased in AP tissue samples compared with controls (p < .05). Positive correlations were observed between the expression of TIMP1 with COMT and CAMK4, TNFA with TIMP1 and CAMK4, COMT with CAMK4. CONCLUSIONS: This study confirms the upregulation of MMP9, TIMP1, TNFA in AP tissues and reports for the first time, the expression of CAMK4 and COMT as suggestive of their involvement in AP pathogenesis.

MicroRNAs Markedly Expressed in Apical Periodontitis Cooperatively Regulate Cytokines and Growth Factors Promoting an Anti-inflammatory Response.

INTRODUCTION: MicroRNAs have been shown to play a role in the pathogenesis of apical periodontitis. Upregulation of miR-10a-5p and downregulation of miR-891a-5p were previously reported in apical periodontitis samples. This study aims to perform a functional characterization of miR-10a-5p, investigating its capacity to regulate the expression of inflammatory cytokines and growth factors, as well as a possible co-regulation mechanism with miR-891a-5p in the development of apical periodontitis. METHODS: miR-10a-5p mimics/controls and miR-891a-5p inhibitors/controls were introduced to human K-562 cells in the presence or absence of lipopolysaccharide. Total RNA was extracted from cell lysates, and target genes were examined via quantitative reverse transcription-polymerase chain reaction. Cell lysates were also subjected to proteomics analysis. Furthermore, mimics of miR-10a-5p and inhibitors of miR-891a-5p were co-transfected into K-562 cells. RNA sequencing and quantitative reverse transcription-polymerase chain reaction were carried out to examine their target genes. RESULTS: Overexpression of miR-10a-5p led to downregulation of tumor necrosis factor-alpha and interleukin-1 beta mRNA and upregulation of transforming growth factor-beta 1 (TGFB1) mRNA expression, whereas interleukin 3 and TGF-ß1 proteins were upregulated. Simultaneous overexpression of miR-10a-5p and inhibition of miR-891a-5p further increased TGFB1 mRNA transcript levels. RNA sequencing revealed that genes co-regulated by miR-10a-5p and miR-891a-5p may be involved in apical periodontitis-related pathways such as tumor necrosis factor, transient receptor potential, and vascular endothelial growth factor signaling pathways. CONCLUSIONS: miR-10a-5p may modulate the expression of multiple inflammatory cytokines and growth factors such as tumor necrosis factor-alpha, IL-1ß, interleukin 3, and TGF-ß1. In addition, miR-10a-5p and miR-891a-5p cooperatively regulate TGFB1 gene expression, and the gene network of this co-regulation is integrated with many pathways in apical periodontitis.

Root Surface Changes in Endodontically Treated Teeth following Orthodontic Movement.

INTRODUCTION: Orthodontically induced external root resorption has been labeled an unavoidable consequence of orthodontic tooth movement (OTM). The objective of this study was to investigate the change in surface area (mm2) and volume (mm3) of endodontically treated teeth (ETT) compared with contralateral teeth with a vital pulp (VPT) after OTM. METHODS: Seventy-six teeth were included in this retrospective analysis: ETT (n = 38) and VPT (n = 38). All teeth were evaluated using cone-beam computed tomographic imaging at 2 time periods: before OTM (T1) and after OTM (T2). Study teeth were segmented to include all areas contained within the lamina dura and then were converted into a mesh model for data calculation. The surface area (mm2) and volume (mm3) of each tooth were calculated at T1 and T2 based on the number of cubic voxels present within the mesh model. Statistical analysis was performed using a linear mixed-effects model. RESULTS: The average change in surface area after OTM in ETT was 13.01 mm2 and 19.95 mm2 in VPT (P < .05). The average percent change in surface area after OTM in ETT was 2.09% and 3.38% in VPT (P < .05). The average change in volume after OTM in ETT was 22.48 mm3 and 32.44 mm3 in VPT (P < .05). The average percent change in volume after OTM in ETT was 2.62% and 4.10% in VPT (P < .05). CONCLUSIONS: The results from this study suggest that ETT are less susceptible to root resorption after OTM than their vital counterparts.

Antibiofilm and immunomodulatory resorbable nanofibrous filing for dental pulp regenerative procedures.

Multifunctional scaffolds with host defense peptides designed for regenerative endodontics are desirable nanobiotechnological tools for dentistry. Here, different scaffolds were tested for use during the pulp revascularization process, including poly(vinyl alcohol)-PVA hydrogels or resins, collagen hydrogels and poly(vinyl alcohol) PVA/Chitosan (PVA/CS) nanofibers. Based on time to degradation (21 days), nanofibers were chosen to be incorporated with ciprofloxacin and IDR-1002 (each at 50 mg/g). Nanofibers containing ciprofloxacin and IDR-1002 had anti-biofilm activity against Enterococcus faecalis, Staphylococcus aureus and a multispecies oral biofilm, besides anti-inflammatory activities. The in vivo subcutaneous tissue response to tooth fragments filled with nanofibers demonstrated a pulp-like tissue formation, when compared to empty teeth fragments. Thus, we designed a strong antimicrobial, immunomodulatory and regenerative candidate for pulp revascularization and regeneration procedures.

Vascular Endothelial Growth Factor and/or Nerve Growth Factor Treatment Induces Expression of Dentinogenic, Neuronal, and Healing Markers in Stem Cells of the Apical Papilla.

INTRODUCTION: The goal of regenerative endodontic procedures is to preserve and stimulate stem cells from the apical papilla (SCAPs) to develop the pulp-dentin complex using various growth factors and scaffolds. We hypothesized that the treatment of SCAPs with vascular endothelial growth factor (VEGF) or nerve growth factor (NGF) may impact the expression of osteogenic and dentinogenic markers. METHODS: The optimum concentration of VEGF and NGF on SCAP viability was assessed and introduced to SCAPs for 6-24 hours. SCAPs were also challenged with Escherichia coli lipopolysaccharide (LPS). Messenger RNA (mRNA) expression of DSPP, DMP1, TGFB1, OCN, SP7, and TWIST1 was examined via quantitative reverse transcription polymerase chain reaction. Immunohistochemistry was used to verify protein expression. In addition, total RNA from NGF-treated SCAPs in the presence or absence of LPS was extracted for RNA sequencing. RESULTS: Compared with untreated cells, NGF-treated SCAPs showed markedly higher levels of DSPP, DMP1, and TGFB1 mRNAs (>9-fold change, P < .05), and SCAPs treated with both VEGF and NGF showed a significant increase of DSPP and TGFB1 mRNAs (P < .05). In addition, in LPS-challenged SCAPs, treatment with these growth factors also exhibited increased expression of DSPP, DMP1, and TGFB1 mRNAs, with the most significant change induced by VEGF (P < .05). Immunohistochemistry confirmed increased dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and transforming growth factor beta 1 protein expression in treated SCAPs. RNA sequencing revealed multiple pathways regulated by NGF, including TGF-ß and neurogenic pathways. CONCLUSIONS: VEGF- and NGF-induced dentinogenic/neuronal/healing marker expression in SCAPs indicates the potential value of applying these growth factors in regenerative endodontic procedures.

Expression Profiling and Functional Characterization of MicroRNAs in Apical Periodontitis.

INTRODUCTION: MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs that may orchestrate the pathogenesis of apical periodontitis (AP). This study aimed to identify differentially expressed miRNAs and investigate their target gene pathways in AP. METHODS: Total RNA was extracted from 10 human AP and 2 healthy apical tissues (controls) and subjected to miRNA sequencing for the identification of differentially expressed miRNAs (>1.5-fold changes). The function of the most up-regulated miRNA was further studied in vitro. miR-10a-5p mimics and inhibitors were introduced to human stem cells from the apical papilla and K-562 cells challenged with lipopolysaccharide, and expressions of predicted target genes were examined via quantitative reverse-transcription polymerase chain reaction and RNA sequencing. RESULTS: A total of 852 miRNAs were identified, of which 12 were significantly up-regulated (1.54- to 8.44-fold) and 94 were significantly down-regulated (0.14- to 0.67-fold) in AP. Predicted target genes of these miRNAs are involved in inflammation, pain, and related pathways. miR-10a-5p showed the highest expression levels in AP. Overexpression of miR-10a-5p in LPS-challenged stem cells from the apical papilla resulted in down-regulation of messenger RNA levels of TNFA and up-regulation of interleukin IL10. RNA sequencing of K-562 cells treated with miR-10a-5p mimics and inhibitors identified miR-10a-5p target genes associated with multiple pathways, including macrophage-mediated inflammation and coagulation pathways. CONCLUSIONS: Over 100 miRNAs were differentially expressed in AP and appeared to be involved with modulation of genes in inflammatory and immune pathways. MiR-10a-5p was the most significantly up-regulated miRNA in AP and may play a critical role in suppressing inflammation and promoting healing.

Genetic and Epigenetic Characterization of Pulpal and Periapical Inflammation.

Pulpal and periapical diseases affect a large segment of the population. The role of microbial infections and host effector molecules in these diseases is well established. However, the interaction between host genes and environmental factors in disease susceptibility and progression is less well understood. Studies of genetic polymorphisms in disease relevant genes have suggested that individual predisposition may contribute to susceptibility to pulpal and periapical diseases. Other studies have explored the contribution of epigenetic mechanisms to these diseases. Ongoing research expands the spectrum of non-coding RNAs in pulpal disease to include viral microRNAs as well. This review summarizes recent advances in the genetic and epigenetic characterization of pulpal and periapical disease, with special emphasis on recent data that address the pathogenesis of irreversible pulpal pathosis and apical periodontitis. Specifically, proinflammatory and anti-inflammatory gene expression and gene polymorphism, as well as recent data on DNA methylation and microRNAs are reviewed. Improved understanding of these mechanisms may aid in disease prevention as well as in improved treatment outcomes.

WNT gene polymorphisms and predisposition to apical periodontitis.

Single nucleotide polymorphisms (SNPs) in WNT genes may impact gene/protein function and contribute to individual predisposition to apical periodontitis (AP). Here, we investigated the association of SNPs in/nearby WNT3, WNT3A, WNT5A, WNT8A, WNT9B and WNT11 genes with AP using a case-control dataset. Cases were defined as individuals with deep caries and AP (n = 188); controls had deep caries and no AP (n = 230). Genotyping was performed using Taqman chemistry in real time PCR. Data analyses was performed using Fisher Exact tests assuming a Bonferroni correction threshold value of 0.005. Single-SNP association analysis revealed a trend for association with WNT3 rs9890413 genotypes (P = 0.009) under a dominant model and allelic association for WNT3A rs1745420 (P = 0.009). Haplotypes involving WNT3-WNT9B-WNT3A alleles were also significantly associated with AP (P ≤ 0.003). Luciferase reporter assays showed higher transcriptional activity (1.4-fold) with the alternate G allele in rs1745420. Expression of WNT3, WNT3A and WNT5A in AP tissues was significantly higher than in control tissues, and inversely correlated with the expression of SERPINB1, COL1A1 and TIMP1 (P < 0.05). Our results suggest that WNT genes have a role in modulating AP and polymorphisms in these genes may increase susceptibility to AP.

Are mTOR and Endoplasmic Reticulum Stress Pathway Genes Associated with Oral and Bone Diseases?

The purpose of this cohort study was to identify associations between combined oral and bone disease phenotypes and genes present in cell regulatory pathways. The studied pathways play important roles in cellular growth, proliferation, differentiation, and homeostasis. DNA samples extracted from whole saliva of 3,912 individuals were genotyped and these data analyzed according to dental caries experience, periapical lesions, periodontitis, osteoporosis, or temporomandibular joint discomfort. Samples were obtained from the Dental Registry and DNA Repository project at the University of Pittsburgh. Twenty-seven polymorphisms in eight genes related to mTOR or endoplasmic reticulum stress pathways were selected for genotyping. Allele frequencies and Hardy-Weinberg equilibrium were calculated. Analyses were performed comparing genotypes between affected and unaffected individuals for each phenotype, as well as for the associated phenotypes combined. For all analyses, we used the software PLINK with an alpha of 0.002. Borderline associations with multiple variants of several genes were found, suggesting that both pathways may be involved in the susceptibility to multiple conditions affecting the oral cavity and bones. When combining patients that had concomitant dental caries, periodontitis, and periapical pathology, several markers in RHEB showed statistically significant association. Multiple conditions affecting bone and teeth (i.e., dental caries, periodontitis, periapical lesion formation, and osteoporosis) appear to share similar underlying genetic etiological factors, which allow us to hypothesize that instead of individually, they should be studied in conjunction in human populations.

PVR/CD155 Ala67Thr Mutation and Cleft Lip/Palate.

The 19q13 locus has been linked to cleft lip and palate by our group and independently by others. Here we fine mapped the region in an attempt to identify an etiological variant that can explain cleft lip and palate occurrence. A total of 2739 individuals born with cleft lip and palate, related to individuals born with cleft lip and palate, and unrelated were studied. We used linkage and association approaches to fine map the interval between D19S714 and D19S433 and genotypes were defined by the use of TaqMan chemistry. We confirmed our previous findings that markers in PVR/CD155 are associated with cleft lip and palate. We studied the mutation Ala67Thr further and calculated its penetrance. We also attempted to detect PVR/CD155 expression in human whole saliva. Our results showed that markers in PVR/CD155 are associated with cleft lip and palate and the penetrance of the Ala67Thr is very low (between 1% and 5%). We could not detect PVR/CD155 expression in adult human whole saliva and PVR/CD155 possibly interacts with maternal infection to predispose children to cleft lip only.

CCR5Δ32 (rs333) polymorphism is associated with decreased risk of chronic and aggressive periodontitis: A case-control analysis based in disease resistance and susceptibility phenotypes.

Chronic and aggressive periodontitis are infectious diseases characterized by the irreversible destruction of periodontal tissues, which is mediated by the host inflammatory immune response triggered by periodontal infection. The chemokine receptor CCR5 play an important role in disease pathogenesis, contributing to pro-inflammatory response and osteoclastogenesis. CCR5Δ32 (rs333) is a loss-of-function mutation in the CCR5 gene, which can potentially modulate the host response and, consequently periodontitis outcome. Thus, we investigated the effect of the CCR5Δ32 mutation over the risk to suffer periodontitis in a cohort of Brazilian patients (total N=699), representative of disease susceptibility (chronic periodontitis, N=197; and aggressive periodontitis, N=91) or resistance (chronic gingivitis, N=193) phenotypes, and healthy subjects (N=218). Additionally, we assayed the influence of CCR5Δ32 in the expression of the biomarkers TNFα, IL-1ß, IL-10, IL-6, IFN-γ and T-bet, and key periodontal pathogens P. gingivalis, T. forsythia, and T. denticola. In the association analysis of resistant versus susceptible subjects, CCR5Δ32 mutant allele-carriers proved significantly protected against chronic (OR 0.49; 95% CI 0.29-0.83; p-value 0.01) and aggressive (OR 0.46; 95% CI 0.22-0.94; p-value 0.03) periodontitis. Further, heterozygous subjects exhibited significantly decreased expression of TNFα in periodontal tissues, pointing to a functional effect of the mutation in periodontal tissues during the progression of the disease. Conversely, no significant changes were observed in the presence or quantity of the periodontal pathogens P. gingivalis, T. forsythia, and T. denticola in the subgingival biofilm that could be attributable to the mutant genotype.

Role of WNT10A in failure of tooth development in humans and zebrafish.

BACKGROUND: Oligodontia is a severe form of tooth agenesis characterized by the absence of six or more permanent teeth. Oligodontia has complex etiology and variations in numerous genes have been suggested as causal for the condition. METHODS: We applied whole-exome sequencing (WES) to identify the cause of oligodontia in a 9-year-old girl missing 11 permanent teeth. Protein modeling and functional analysis in zebrafish were also performed to understand the impact of identified variants on the phenotype. RESULTS: We identified a novel compound heterozygous missense mutation in WNT10A (c.637G>A:p.Gly213Ser and c.1070C>T:p.Thr357Ile) as the likely cause of autosomal recessive oligodontia in the child. Affected residues are located in conserved regions and variants are predicted to be highly deleterious for potentially destabilizing the protein fold and inhibiting normal protein function. Functional studies in zebrafish embryos showed that wnt10a is expressed in the craniofacies at critical time points for tooth development, and that perturbations of wnt10a expression impaired normal tooth development and arrested tooth development at 5 days postfertilization (dpf). Furthermore, mRNA expression levels of additional tooth development genes were directly correlated with wnt10a expression; expression of msx1, dlx2b, eda, and axin2 was decreased upon wnt10a knockdown, and increased upon wnt10a overexpression. CONCLUSIONS: Our results reveal a novel compound heterozygous variant in WNT10A as pathogenic for oligodontia, and demonstrate that perturbations of wnt10a expression in zebrafish may directly and/or indirectly affect tooth development recapitulating the agenesis phenotype observed in humans.

A possible relationship between gluconeogenesis and glycogen metabolism in rabbits during myocardial ischemia.

Ischemia is responsible for many metabolic abnormalities in the heart, causing changes in organ function. One of modifications occurring in the ischemic cell is changing from aerobic to anaerobic metabolism. This change causes the predominance of the use of carbohydrates as an energy substrate instead of lipids. In this case, the glycogen is essential to the maintenance of heart energy intake, being an important reserve to resist the stress caused by hypoxia, using glycolysis and lactic acid fermentation. In order to study the glucose anaerobic pathways utilization and understand the metabolic adaptations, New Zealand white rabbits were subjected to ischemia caused by Inflow occlusion technique. The animals were monitored during surgery by pH and lactate levels. Transcription analysis of the pyruvate kinase, lactate dehydrogenase and phosphoenolpyruvate carboxykinase enzymes were performed by qRT-PCR, and glycogen quantification was determined enzymatically. Pyruvate kinase transcription increased during ischemia, followed by glycogen consumption content. The gluconeogenesis increased in control and ischemia moments, suggesting a relationship between gluconeogenesis and glycogen metabolism. This result shows the significant contribution of these substrates in the organ energy supply and demonstrates the capacity of the heart to adapt the metabolism after this injury, sustaining the homeostasis during short-term myocardial ischemia.

A possible relationship between gluconeogenesis and glycogen metabolism in rabbits during myocardial ischemia

ABSTRACT Ischemia is responsible for many metabolic abnormalities in the heart, causing changes in organ function. One of modifications occurring in the ischemic cell is changing from aerobic to anaerobic metabolism. This change causes the predominance of the use of carbohydrates as an energy substrate instead of lipids. In this case, the glycogen is essential to the maintenance of heart energy intake, being an important reserve to resist the stress caused by hypoxia, using glycolysis and lactic acid fermentation. In order to study the glucose anaerobic pathways utilization and understand the metabolic adaptations, New Zealand white rabbits were subjected to ischemia caused by Inflow occlusion technique. The animals were monitored during surgery by pH and lactate levels. Transcription analysis of the pyruvate kinase, lactate dehydrogenase and phosphoenolpyruvate carboxykinase enzymes were performed by qRT-PCR, and glycogen quantification was determined enzymatically. Pyruvate kinase transcription increased during ischemia, followed by glycogen consumption content. The gluconeogenesis increased in control and ischemia moments, suggesting a relationship between gluconeogenesis and glycogen metabolism. This result shows the significant contribution of these substrates in the organ energy supply and demonstrates the capacity of the heart to adapt the metabolism after this injury, sustaining the homeostasis during short-term myocardial ischemia.

Heat Shock 70 Protein Genes and Genetic Susceptibility to Apical Periodontitis.

INTRODUCTION: Heat shock proteins (HSPs) protect cells under adverse conditions such as infection, inflammation, and disease. The differential expression of HSPs in human periapical granulomas suggests a potential role for these proteins in periapical lesion development, which may contribute to different clinical outcomes. Therefore, we hypothesized that polymorphisms in HSP genes leading to perturbed gene expression and protein function may contribute to an individual's susceptibility to periapical lesion development. METHODS: Subjects with deep carious lesions with or without periapical lesions (≥3 mm) were recruited at the University of Texas School of Dentistry at Houston and at the University of Pittsburgh. Genomic DNA samples of 400 patients were sorted into 2 groups: 183 cases with deep carious lesions and periapical lesions (cases) and 217 cases with deep carious lesions but without periapical lesions (controls). Eight single nucleotide polymorphisms (SNPs) in HSPA4, HSPA6, HSPA1L, HSPA4L, and HSPA9 genes were selected for genotyping. Genotypes were generated by end point analysis by using Taqman chemistry in a real-time polymerase chain reaction assay. Allele and genotype frequencies were compared among cases and controls by using χ(2) and Fisher exact tests as implemented in PLINK v.1.07. In silico analysis of SNP function was performed by using Polymorphism Phenotyping V2 and MirSNP software. RESULTS: Overall, SNPs in HSPA1L and HSPA6 showed significant allelic association with cases of deep caries and periapical lesions (P < .05). We also observed altered transmission of HSPA1L SNP haplotypes (P = .03). In silico analysis of HSPA1L rs2075800 function showed that this SNP results in a glutamine-to-lysine substitution at position 602 of the protein and might affect the stability and function of the final protein. CONCLUSIONS: Variations in HSPA1L and HSPA6 may be associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing apical periodontitis.

Evaluating the effects of different dental devices on implantable cardioverter defibrillators.

INTRODUCTION: The implantable cardioverter defibrillator (ICD) is an electronic device that emits electrical signals to the heart via lead wires and electrodes. It is used for cardiac rhythm monitoring and treatment. Because electronic dental devices have been shown to produce electromagnetic fields, we hypothesize that they may interfere with ICD function. METHODS: Nine dental devices (heat carrier, electronic apex locator, electric pulp tester, unipolar electrosurgery unit, electric motor, curing light, and 3 gutta-percha guns) were tested in this study for their ability to interfere with the function of 4 ICDs (2 single-chambered and 2 dual-chambered ICDs). ICD activity was monitored for 30 seconds using an ICD programmer (Medtronic 2090; Minneapolis, MN) and evaluated through an electrogram test strip printout. RESULTS: Electromagnetic interference was detected with the electric motor, curing light, electric pulp tester, and electrosurgery unit although no electromagnetic disturbances were detected with these devices. No electromagnetic interferences were observed for the gutta-percha guns, heat carrier, and apex locator. However, the electrosurgery unit affected the dual-chambered ICD (Consulta CRT-D, Medtronic) and delivered therapies for fibrillation when no ventricular fibrillation was present. CONCLUSIONS: Our results suggest that the electrosurgery unit produces electromagnetic disturbances with unwanted therapy delivery shock and potentially clinically significant outcomes.

FOXP3 DNA methylation levels as a potential biomarker in the development of periapical lesions.

INTRODUCTION: Epigenetic mechanisms, such as DNA methylation, can modify gene expression patterns without changing the DNA sequence, comprising a tool that cells use to lock genes in the "off" position. Variations in the methylation profile have been correlated to a variety of human diseases. Here, we hypothesize that DNA methylation in immune response-related genes may contribute to the development of periapical lesions. METHODS: The DNA methylation patterns of 22 immune response-related gene promoters were evaluated in 137 human periapical granulomas, 8 apical cysts, and 31 healthy gingival tissues from 2 independent cohorts using a pathway-specific real-time polymerase chain reaction array (EpiTect Methyl II; Qiagen Inc, Valencia, CA). Messenger RNA expression analysis by qualitative polymerase chain reaction was also performed. SABiosciences's hierarchical clustering and methylation (Qiagen, Valencia, CA) and Prism6 software (GraphPad Software, Inc, La Jolla, CA) were used for data analysis. RESULTS: FOXP3 gene promoter showed the highest level of methylation in both periapical granulomas and apical cysts (P < .001), and methylation levels were inversely correlated with FOXP3 messenger RNA expression in the lesions. Furthermore, FOXP3 expression was prevalent in inactive lesions and was positively correlated with interleukin-10 and transforming growth factor beta levels. CONCLUSIONS: Our results suggest that FOXP3 acts as a master switch governing the development and function of T-regulatory cells, whose functions include the inhibition of immune responses and temper inflammation. The observed differential methylation patterns of FOXP3 in periapical lesions may be crucial in determining its suppressive activity and may be involved in periapical lesion development.

Association of WNT9B Gene Polymorphisms With Nonsyndromic Cleft Lip With or Without Cleft Palate in Brazilian Nuclear Families.

OBJECTIVE: Nonsyndromic cleft lip with or without cleft palate (NSCL±P) is a common craniofacial anomaly of complex etiology in people. WNT pathway genes have important roles during craniofacial development, and an association of WNT genes with NSCL±P has been demonstrated in different populations. The aim of this study was to evaluate the association between polymorphisms in WNT3 and WNT9B genes and CL/P in Brazilian families. PATIENTS: Seventy nuclear families composed of an affected child and the child's unaffected parents were examined clinically. Saliva samples were collected for molecular analyses. DESIGN: Three single nucleotide polymorphisms (SNPs) in the WNT3 gene and two in WNT9B were investigated in real-time polymerase chain reaction using TaqMan chemistry. The Family-Based Association Test and the transmission disequilibrium test were used to verify the association between each marker allele and NSCL±P. The level of significance was established at P ≤ .01 after Bonferroni correction. RESULTS: A positive association was detected between NSCL±P and SNP rs1530364 in the WNT9B gene. Haplotype analysis showed an association of WNT3 and WNT9B haplotypes. No association was detected between NSCL±P and individual SNPs in WNT3. CONCLUSION: Our study further supports the involvement of WNT9B as a cleft susceptibility gene in Brazilian families experiencing NSCL±P. Although additional studies are still necessary to unveil the exact mechanism by which WNT genes would contribute to NSCL±P, allelic polymorphisms in these genes and their interactions may partly explain the variance of individual susceptibility to NSCL±P.

Role of TRAV locus in low caries experience.

Caries is the most common chronic, multifactorial disease in the world today; and little is still known about the genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified five loci related to caries susceptibility: 5q13.3, 13q31.1, 14q11.2, 14q 24.3, and Xq27. In the present study, we fine mapped the 14q11.2 locus to identify genetic contributors to caries susceptibility. Four hundred seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. An additional 387 DNA samples from unrelated individuals were used to determine allele frequencies. For replication purposes, a total of 1,446 independent subjects from four different populations were analyzed based on their caries experience (low versus high). Forty-eight markers in 14q11.2 were genotyped using TaqMan chemistry. Transmission disequilibrium test was used to detect over transmission of alleles in the Filipino families, and Chi-square, Fisher's exact and logistic regression were used to test for association between low caries experience and variant alleles in the replication data sets. We finally assessed the mRNA expression of TRAV4 in the saliva of 143 study subjects. In the Filipino families, statistically significant associations were found between low caries experience and markers in TRAV4. We were able to replicate these results in the populations studied that were characteristically from underserved areas. Direct sequencing of 22 subjects carrying the associated alleles detects one missense mutation (Y30R) that is predicted to be probably damaging. Finally, we observed higher expression in children and teenagers with low caries experience, correlating with specific alleles in TRAV4. Our results suggest that TRAV4 may have a role in protecting against caries.

Further evidence of association of the ABCA4 gene with cleft lip/palate.

Non-syndromic cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex etiology. Numerous genes and environmental factors, and their interactions, are thought to play a role in the susceptibility to CL/P. A recent genome-wide association study with several populations revealed markers in/near transcription factor vmaf musculoaponeurtoic fibrosarcoma oncogene homolog B (MAFB) and ATP-binding cassette sub-family A member 4 (ABCA4) genes as new susceptibility loci for CL/P. We hypothesized that these genes could also contribute to CL/P in a Brazilian population, and hence we evaluated if the associated single-nucleotide polymorphisms (SNPs) in MAFB (rs13041247 and rs11696257) and ABCA4 (rs560426 and rs481931) were associated with CL/P in our case-control data set. We genotyped 812 Caucasian individuals (400 cases and 412 controls) from Brazil. Allele frequencies were compared for cases and controls as well as for cleft subgroups and controls. ABCA4 rs540426 showed strong associations with CL/P, unilateral and right CL/P, and bilateral CL/P, whereas the SNP rs481931 showed borderline associations with CL/P and bilateral CL/P . No association was found for MAFB. Our results support a potential role for ABCA4 in the etiology of CL/P in individuals from Brazil.