Avaliação da capacidade carcinogênica do extrato aquoso de café, em células somáticas de Drosophila melanogaster / Assessment of the carcinogenic capacity of commercial aqueous extract of coffea arabica in somatic cells of Drosophila melanogaster

Objetivo: devido ao consumo rotineiro do café pela população brasileira, o presente trabalho tem como objetivo avaliar o potencial carcinogênico de diferentes concentrações do café (Coffea arabica), por meio do Teste para Detecção de Tumor Epitelial em Drosophila melanogaster. Métodos: para avaliar o efeito carcinogênico do café, larvas de 3° estágio descendentes do cruzamento entre fêmeas virgens wts/TM3, sb¹ e machos mwh/mwh foram tratadas com diferentes concentrações de café comercial (100; 50; 25; 12,5 e 6,26 mg/mL). Após a exposição (48h) e o processo de metamorfose, as moscas adultas foram analisadas quanto à presença de tumor epitelial, e os grupos tratados foram comparados com o controle negativo (água ultrapura). A toxicidade do café foi mensurada por meio da taxa de moscas que sobreviveram a etapa de metamorfose após exposição. Resultados: foi observado diferença estatisticamente significativa na taxa de sobrevivência (p < 0,05) e na frequência de tumor epitelial total (p < 0,05) em moscas tratadas com 100 mg/mL de café, quando comparado ao controle negativo. Conclusões: o café, na concentração de 100 mg/mL, foi tóxico e carcinogênico para D. melanogaster.

Objective: Due to the routine consumption of coffee by the Brazilian population, the present work aims to evaluate the carcinogenic potential of different concentrations of coffee (Coffea arabica) through the Test for Epithelial Tumor Detection in Drosophila melanogaster. Methods: In order to evaluate the carcinogenic effect of coffee, third-stage larvae descended from the cross between virgin females wts/TM3, sb1 and males mwh/mwh were treated with different concentrations of commercial coffee (100, 50, 25, 12, 26 mg/mL). After exposure (48h) and the metamorphosis process, the adult flies were analyzed for the presence of an epithelial tumor and the treated groups were compared with the negative control (ultrapure water). Coffee toxicity was measured by the rate of flies that survived the post-exposure metamorphosis stage. Results: A statistically significant difference was observed in survival rate (p < 0.05) and frequency of total epithelial tumor (p < 0.05) in flies treated with 100 mg/mL of coffee, when compared to the negative control. Conclusions: Coffee at the concentration of 100 mg/mL was toxic and carcinogenic to D. melanogaster.

miRNA Genetic Variants Alter Their Secondary Structure and Expression in Patients With RASopathies Syndromes.

RASopathies are a group of rare genetic diseases caused by germline mutations in genes involved in the RAS-mitogen-activated protein kinase (RAS-MAPK) pathway. Whole-exome sequencing (WES) is a powerful approach for identifying new variants in coding and noncoding DNA sequences, including miRNAs. miRNAs are fine-tuning negative regulators of gene expression. The presence of variants in miRNAs could lead to malfunctions of regulation, resulting in diseases. Here, we identified 41 variants in mature miRNAs through WES analysis in five patients with previous clinical diagnosis of RASopathies syndromes. The pathways, biological processes, and diseases that were over-represented among the target genes of the mature miRNAs harboring variants included the RAS, MAPK, RAP1, and PIK3-Akt signaling pathways, neuronal differentiation, neurogenesis and nervous system development, congenital cardiac defects (hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy), and the phenotypes and syndromes of RASopathies (Noonan syndrome, Legius syndrome, Costello syndrome, Cafe au lait spots multiple, subaortic stenosis, pulmonary valve stenosis, and LEOPARD syndrome). Furthermore, eight selected variants in nine mature miRNAs (hsa-miR-1304, hsa-miR-146a, hsa-miR-196a2, hsa-miR-499a/hsa-miR-499b, hsa-miR-449b, hsa-miR-548l, hsa-miR-575, and hsa-miR-593) may have caused alterations in the secondary structures of miRNA precursor. Selected miRNAs containing variants such as hsa-miR-146a-3p, hsa-miR-196a-3p, hsa-miR-548l, hsa-miR-449b-5p, hsa-miR-575, and hsa-miR499a-3p could regulate classical genes associated with Rasopathies and RAS-MAPK pathways, contributing to modify the expression pattern of miRNAs in patients. RT-qPCR expression analysis revealed four differentially expressed miRNAs that were downregulated: miRNA-146a-3p in P1, P2, P3, P4, and P5, miR-1304-3p in P2, P3, P4, and P5, miR-196a2-3p in P3, and miR-499b-5p in P1. miR-499a-3p was upregulated in P1, P3, and P5. These results indicate that miRNAs show different expression patterns when these variants are present in patients. Therefore, this study characterized the role of miRNAs harboring variants related to RASopathies for the first time and indicated the possible implications of these variants for phenotypes of RASopathies such as congenital cardiac defects and cardio-cerebrovascular diseases. The expression and existence of miRNA variants may be used in the study of biomarkers of the RASopathies.

Estudo descritivo molecular de pacientes com RASopatia / Descriptive molecular study of patients with RASopathy

As RASopatias são um grupo de doenças cujos pacientes apresentam alterações constitucionais em genes que participam de uma mesma via de sinalização celular denominada Ras/MAPK, que desempenha um papel importante na proliferação, diferenciação, migração celular e apoptose, além de estar associada a processos carcinogênicos. Apesar dos avanços em métodos diagnósticos, cerca de 20 a 25% dos casos permanecem inconclusivos, o que impulsiona pesquisas que buscam alterações em outros níveis de regulação da via, como RNAs não codificantes e proteínas. O primeiro capítulo deste estudo, avalia um grupo de 6 pacientes diagnosticados clinicamente com RASopatia e com exomas sequenciados. Foram obtidos dados sobre mutações em seus miRNAs. O segundo capítulo relata o caso de um paciente com suspeita clínica de síndrome de Costello, mas sem mutações detectadas. Foram avaliados, in silico, dados sobre miRNAs reguladores do gene HRAS, bem como os diferentes tecidos nos quais o HRAS é expresso. Para o capítulo 3 foi realizado um estudo comparativo entre gêmeas com diagnóstico clínico de síndrome de Noonan, mas com fenótipo discordante. Foi realizado um array por RT-qPCR para diferentes RNAs reguladores e um estudo comparativo de proteoma com análise de vias biológicas, processos biológicos e genes alvo da regulação de fatores de transcrição putativos. No estudo de miRNAs foram encontradas mutações em heterozigose, que são de difícil avaliação em ensaios de expressão. No estudo de caso do paciente S4 (síndrome de Costello), não foram encontradas mutações nos miR-181d-5p, let-7a-5p, miR-143-3p, miR-181a-5p, miR-139-5p, miR-663a e let-7b-5p, descritos como reguladores do HRAS. Também não foram encontrados tecidos viáveis para coleta e análise da expressão do HRAS. Na expressão de RNAs reguladores nas gêmeas (S16 e S17) foram encontrados níveis de expressão aumentados em S17 para Lnc-C21orf33-1, ERBS3/SBNO2e, miR-200be, CTBP1-AS e Lnc_DC. Na análise do proteoma, foram encontradas diferenças de expressão em vias de integrinas, proteoglicanos e trombinas, além de diferenças em processos de transdução de sinal, crescimento e manutenção celular e metabolismo. Os genes com sítio de ligação para os fatores de transcrição como RREB1, ETS1, EGR1 e TBX5 também possuíam expressão diferente entre as gêmeas. Os resultados aqui apresentados apontam novos caminhos para estudos moleculares das RASopatias que possam preencher as lacunas diagnósticas ainda pendentes.

RASopathies are a group of diseases whose patients present constitutional changes in genes that participate in the same cellular signaling pathway called the Ras/MAPK, which plays an important role in proliferation, differentiation, cell migration and apoptosis, in addition to being associated with carcinogenic processes. Despite advances in diagnostic methods, about 20 to 25% of cases remain inconclusive, which drives research that seeks changes in other levels of pathway regulation, such as non-coding RNAs and proteins. The first chapter of this study evaluates a group of 6 patients clinically diagnosed with RASopathy and sequenced exomes. Data were obtained on mutations in their miRNAs. The second chapter reports the case of a patient with clinical suspicion of Costello syndrome, but without mutations detected. Data on the miRNAs regulating the HRAS gene, as well as the different tissues in which HRAS is expressed, were evaluated in silico. For chapter 3 a comparative study was performed between twins with clinical diagnosis of Noonan syndrome, but with a discordant phenotype. An array was performed by RT-qPCR for different regulatory RNAs and a comparative proteome study with analysis of biological pathways, biological processes and genes targeting the regulation of putative transcription factors. In the study of miRNAs, mutations were found in heterozygosis, which are difficult to evaluate in expression assays. In the case study of S4 patient (Costello syndrome), no mutations were found in miR-181d-5p, let-7a-5p, miR-143-3p, miR-181a-5p, miR-139-5p, miR-663a and let-7b -5p, described as HRAS regulators. No available tissues were also found for collection and analysis of HRAS expression. In expression of regulatory RNAs in the S16 and S17 twins, increased levels of S17 expression were found for Lnc-C21orf33-1, ERBS3 / SBNO2e, miR-200b, CTBP1-AS and Lnc_DC. In the proteome analysis, expression differences were found in integrins, proteoglycans and thrombin pathways, as well as differences in signal transduction processes, cell growth and maintenance, and metabolism. Genes with binding site for transcription factors such as RREB1, ETS1, EGR1 and TBX5 also had different expression between the twins. The results presented here point out new ways for molecular studies of RASopathies that may close the remaining diagnostic gaps.

Chronic Periodontitis and RANKL/OPG Ratio in Peri-Implant Mucosae Inflammation

Abstract tHistory of chronic periodontitis (CP) is a risk factor for oseointegration failure. The osteoclastogenesis system (RANK, RANKL and OPG) is critical for bone homeostatic control. We investigated the levels of OPG and RANKL in peri-implant tissues from volunteers with and without a history of CP and their association with mucosae inflammation. This is a single-blind case-contro study. Diagnosis of a history of CP and peri-implant examination was performed on 46 volunteers, divided into control (without history of CP, n=26) and CP group (with history of CP, n=20). Gingival biopsies were harvested during implant exposure. Quantitative PCR evaluated OPG/RANKL mRNA expressions. OPG and RANKL proteins were analyzed by western blot and immunohistochemistry assay. The chi-square test analyzed the significance of nominal variables between groups while continuous variables were analyzed by T-test or Mann-Whitney test, after Shapiro-Wilk test evaluation. The 2-ΔΔCT Livak method calculation evaluated the gene expression. Values of p<0.05 were considered statistically significant. Volunteers with CP history had 23 times higher chance of developing mucosae inflammation. High mucosae levels of RANKL (p=0.04) and RANKL/OPG (p=0.001) mRNA expressions were observed in CP group. CP volunteers showed increased RANKL protein levels in opposition to decreased OPG expression. Even without active periodontitis, volunteers with a history of CP had elevated gingival levels of RANKL/OPG and higher correlation with peri-implant mucosae inflammation and implant loss.

Resumo A história de periodontite crônica (CP) é um fator de risco para falhas na osseointegração. O sistema de osteoclastogênese (RANK, RANKL e OPG) é crucial para o controle da homeostase óssea. O objetivo deste estudo foi investigar os níveis de OPG e RANKL no tecido peri-implantar de voluntários com e sem histórico de CP e sua associação com inflamação da mucosa. Este é um estudo tipo caso-controle. O exame para diagnóstico de CP e na região peri-implantar foi realizado em 46 voluntários, divididos em controle (sem história CP, n=26) e grupo CP (com histórico de CP, n=20). Descartes gengivais foram obtidos durante a exposição do implante. PCR quantitativo avaliou a expressão do RNAm de OPG/RANKL. As proteínas OPG e RANKL foram analisadas por western blot e imunohistoquímica. O teste do qui-quadrado analisou a significância entre as variáveis nominais enquanto as variáveis contínuas foram analisadas pelo teste-t e Mann-Whitney, após o teste de Shapiro-wilk. O método do Livak 2--ΔΔCT avaliou a expressão gênica. Os voluntários com CP apresentaram 23 vezes mais chances de desenvolver inflamação da mucosa. Expressão elevada no RNAm de RANKL (p=0.04) e RANKL/OPG (p=0.001) foram observadas no grupo CP. Voluntários com CP mostraram aumento dos níveis da proteína RANKL em contraste com diminuída expressão de OPG. Mesmo sem periodontite ativa, voluntários com histórico de CP apresentaram elevado nível gengival de RANKL/OPG e alta correlação com inflamação peri-implantar e perda do implante.

Chronic Periodontitis and RANKL/OPG Ratio in Peri-Implant Mucosae Inflammation.

tHistory of chronic periodontitis (CP) is a risk factor for oseointegration failure. The osteoclastogenesis system (RANK, RANKL and OPG) is critical for bone homeostatic control. We investigated the levels of OPG and RANKL in peri-implant tissues from volunteers with and without a history of CP and their association with mucosae inflammation. This is a single-blind case-contro study. Diagnosis of a history of CP and peri-implant examination was performed on 46 volunteers, divided into control (without history of CP, n=26) and CP group (with history of CP, n=20). Gingival biopsies were harvested during implant exposure. Quantitative PCR evaluated OPG/RANKL mRNA expressions. OPG and RANKL proteins were analyzed by western blot and immunohistochemistry assay. The chi-square test analyzed the significance of nominal variables between groups while continuous variables were analyzed by T-test or Mann-Whitney test, after Shapiro-Wilk test evaluation. The 2-ΔΔCT Livak method calculation evaluated the gene expression. Values of p<0.05 were considered statistically significant. Volunteers with CP history had 23 times higher chance of developing mucosae inflammation. High mucosae levels of RANKL (p=0.04) and RANKL/OPG (p=0.001) mRNA expressions were observed in CP group. CP volunteers showed increased RANKL protein levels in opposition to decreased OPG expression. Even without active periodontitis, volunteers with a history of CP had elevated gingival levels of RANKL/OPG and higher correlation with peri-implant mucosae inflammation and implant loss.

Assessment of carcinogenic potential of soft drinks of cola, diet cola, orange and lemon, produced in the city of Uberlândia, Minas Gerais state, Brazil / Avaliação do potencial carcinogênico de refrigerantes à base de cola, diet cola, laranja e limão, produzidos no município de Uberlândia, estado de Minas Gerais, Brasil

Soft drinks are industrialized unfermented beverages, free of alcohol, carbonated, rich in artificial flavors and sugar. The intense consumption of such beverages can be related to not inheritable diseases such as caries, allergy, cellulite and stretch marks, gastrointestinal disorders, diabetes and cancer. The aim of this study was to evaluate the carcinogenic potential of different concentrations of soft drinks produced in the Uberlândia city, Minas Gerais State, Brazil, by means of Epithelial Tumor Detection Test using Drosophila melanogaster as a model. Third stage larvae descendants of crosses between D. melanogaster virgin females wts/TM3, sb¹ and males mwh/mwh were treated with different concentrations (0.83, 1.66 or 3.33 mL/g) of cola, diet cola, orange or lemon soft drinks. The total epithelial tumor rate observed in flies treated with 3.33 mL/g of cola and orange soft drinks was higher than the negative control. The diet cola and lemon caused no significant increase in the overall frequency of epithelial tumors in D. melanogaster. In conclusion, in these experimental conditions, the cola and orange base soft drinks demonstrated carcinogenic potential in somatic cells of D. melanogaster in the concentration of 3.33 mL/g.

Refrigerantes são bebidas industrializadas não fermentadas, livre de álcool, carbonatadas, ricas em aromas artificiais e açúcar. O consumo intenso dessas bebidas pode estar relacionada à doenças não herdáveis como, cáries, quadros alérgicos, formação de celulite e estrias cutâneas, alterações gastrointestinais, diabetes e câncer. O objetivo desse trabalho foi avaliar o potencial carcinogênico de diferentes concentrações de refrigerantes produzidos no município de Uberlândia, Estado de Minas Gerais, Brasil. Foi realizado o Teste de Detecção de Tumor Epitelial em Drosophila melanogaster. Larvas de terceiro estágio descendentes do cruzamento entre fêmeas virgens wts/TM3, sb¹ e machos mwh/mwh de D. melanogaster foram tratadas com diferentes concentrações (0,83; 1,66 ou 3,33 mL/g) de refrigerantes à base de cola, diet cola, laranja e limão. Os resultados mostraram aumento na frequência de tumor epitelial em moscas tratadas 3,33 mL/g de refrigerantes à base de cola e de laranja, quando comparados ao controle negativo. Os refrigerantes diet cola e limão não provocaram aumento na frequência de tumor epitelial em D. melanogaster. Em conclusão, nessas condições experimentais, os refrigerantes à base de cola e laranja mostraram potencial carcinogênico em células somáticas de D. melanogaster na concentração de 3,33 mL/g.

Genetic Variants in Folate and Cobalamin Metabolism-Related Genes in Nonsyndromic Cleft Lip and/or Palate

The aim of this study was to evaluate the association of the polymorphisms in TCN2 (rs1801198) gene and in MTRR (rs1801394) gene with nonsyndromic cleft lip and/or palate (NSCL/P) in a Brazilian population. Genomic DNA was extracted from buccal cells. The polymorphisms in TCN2 (rs1801198) and MTRR (rs1801394) genes were genotyped by carrying out real-time PCR and Taqman assay. Chi-square test was used to determine the association between genotype and allele frequencies with NSCL/P and NSCL/P subgroups (cleft lip only, cleft lip and palate, and cleft palate only). Eight hundred and sixty seven unrelated individuals (401 cases with NSCL/P and 466 individuals without cleft) were evaluated. Genotype distributions of TCN2 and MTRR polymorphisms were in Hardy-Weinberg equilibrium. The TCN2 polymorphic genotype GG was identified in 16.7% of the NSCL/P group and in 14.1% of the non-cleft group (p>0.05). Similarly, the frequency of MTRR genotype (GG) was similar in NSCL/P group (15.5%) and control group (17.8%) (p>0.05). Multivariate analysis showed an association between MTRR and the subgroup that the mother smoked during pregnancy (p=0.039). Our findings did not demonstrate an association between TCN2 polymorphisms and NSCL/P, however suggests an association between MTRR and NSCL/P etiology.

Resumo O objetivo desse estudo foi avaliar a associação entre os polimorfismos no gene TCN2 (rs1801198) e no gene MTRR (rs1801394) com fissura de lábio e/ou palato não sindrômica (NSFL/P) em uma população brasileira. DNA genômico foi extraído de células bucais. Os polimorfismos nos genes TCN2 (rs1801198) e MTRR (rs1801394) foram genotipados através do PCR em tempo real pelo método Taqman. O teste do qui-quadrado foi utilizado para determinar a associação entre a frequência alélica e genotípica e NSFL/P e nos subtipos (fissura de lábio, fissura de lábio com palato e fissura de palato). Oitocentos e sessenta e sete indivíduos não aparentados (401 casos com NSFL/P e 466 indivíduos sem fissura) foram avaliados. A distribuição dos genótipos dos polimorfismos de TCN2 e MTRR estavam em equilíbrio de Hardy-Weinberg. O genótipo polimórfico GG do gene TCN2 foi identificado em 16,7% do grupo com NSFL/P e em 14,1% do grupo sem fissura (p>0,05). Da mesma forma, a freqüência do genótipo GG do gene MTRR foi bastante semelhante entre o grupo com NSFL/P (15,5%) e o grupo controle (17,8%). A análise multivariada mostrou associação entre o gene MTRR e o subgrupo que apresentou tabagismo materno durante a gestação (p=0,039). Nossos resultados mostraram que não há associação entre os polimorfismos nos genes TCN2 e NSFL/P, entretanto sugerem uma associação entre MTRR e a etiologia de NSFL/P.

Different contribution of BRINP3 gene in chronic periodontitis and peri-implantitis: a cross-sectional study.

BACKGROUND: Peri-implantitis is a chronic inflammation, resulting in loss of supporting bone around implants. Chronic periodontitis is a risk indicator for implant failure. Both diseases have a common etiology regarding inflammatory destructive response. BRINP3 gene is associated with aggressive periodontitis. However, is still unclear if chronic periodontitis and peri-implantitis have the same genetic background. The aim of this work was to investigate the association between BRINP3 genetic variation (rs1342913 and rs1935881) and expression and susceptibility to both diseases. METHODS: Periodontal and peri-implant examinations were performed in 215 subjects, divided into: healthy (without chronic periodontitis and peri-implantitis, n = 93); diseased (with chronic periodontitis and peri-implantitis, n = 52); chronic periodontitis only (n = 36), and peri-implantitis only (n = 34). A replication sample of 92 subjects who lost implants and 185 subjects successfully treated with implants were tested. DNA was extracted from buccal cells. Two genetic markers of BRINP3 (rs1342913 and rs1935881) were genotyped using TaqMan chemistry. Chi-square (p < 0.05) compared genotype and allele frequency between groups. A subset of subjects (n = 31) had gingival biopsies harvested. The BRINP3 mRNA levels were studied by CT method (2(ΔΔCT)). Mann-Whitney test correlated the levels of BRINP3 in each group (p < 0.05). RESULTS: Statistically significant association between BRINP3 rs1342913 and peri-implantitis was found in both studied groups (p = 0.04). The levels of BRINP3 mRNA were significantly higher in diseased subjects compared to healthy individuals (p = 0.01). CONCLUSION: This study provides evidence that the BRINP3 polymorphic variant rs1342913 and low level of BRINP3 expression are associated with peri-implantitis, independently from the presence of chronic periodontitis.

Genetic Variants in Folate and Cobalamin Metabolism-Related Genes in Nonsyndromic Cleft Lip and/or Palate.

The aim of this study was to evaluate the association of the polymorphisms in TCN2 (rs1801198) gene and in MTRR (rs1801394) gene with nonsyndromic cleft lip and/or palate (NSCL/P) in a Brazilian population. Genomic DNA was extracted from buccal cells. The polymorphisms in TCN2 (rs1801198) and MTRR (rs1801394) genes were genotyped by carrying out real-time PCR and Taqman assay. Chi-square test was used to determine the association between genotype and allele frequencies with NSCL/P and NSCL/P subgroups (cleft lip only, cleft lip and palate, and cleft palate only). Eight hundred and sixty seven unrelated individuals (401 cases with NSCL/P and 466 individuals without cleft) were evaluated. Genotype distributions of TCN2 and MTRR polymorphisms were in Hardy-Weinberg equilibrium. The TCN2 polymorphic genotype GG was identified in 16.7% of the NSCL/P group and in 14.1% of the non-cleft group (p>0.05). Similarly, the frequency of MTRR genotype (GG) was similar in NSCL/P group (15.5%) and control group (17.8%) (p>0.05). Multivariate analysis showed an association between MTRR and the subgroup that the mother smoked during pregnancy (p=0.039). Our findings did not demonstrate an association between TCN2 polymorphisms and NSCL/P, however suggests an association between MTRR and NSCL/P etiology.

Studies of genes involved in craniofacial development and tumorigenesis: FGF3 contributes to isolated oral clefts and may interact with PAX9.

OBJECTIVE: Previous studies suggest individuals born with oral clefts and their families have a higher susceptibility for cancer, which raises the hypothesis that these two conditions share common molecular pathways. This study evaluated the association between oral clefts and polymorphisms in genes that play a role in craniofacial and tumor development. MATERIALS AND METHODS: Four hundred and ninety-seven subjects born with oral clefts and 823 unaffected subjects were recruited. Twenty-nine markers in 13 genes were genotyped by the Taqman method. Chi-square was used to compare allele and genotype frequencies. Bonferroni correction for multiple testing was used and the established alpha was 0.0003. This study also used logistic regression to test if genetic variants were associated with oral clefts using positive family history of cancer and age as covariates. RESULTS: There was no association between family history of cancer and oral clefts (p = 0.51). None of the 1320 study participants had a diagnosis of cancer at the time of participation in the study. The marker rs4980700 in FGF3 was associated with oral clefts (p = 0.0002). Logistic regression analysis also provided evidence for gene-gene interaction between FGF3 (rs4980700) and PAX9 (rs2073242), increasing the risk for isolated oral clefts (p = 0.0003). CONCLUSION: FGF3 is associated with oral clefts and may interact with PAX9.

Evidence of genetic variations associated with rotator cuff disease.

BACKGROUND: Rotator cuff disease (RCD) is a complex process influenced by a multitude of factors, and a number of gene pathways are altered in rotator cuff tears. Polymorphisms in these genes can lead to an extended tendon degeneration process, which explains why subsets of patients are more susceptible to RCD. MATERIALS AND METHODS: Twenty-three single-nucleotide polymorphisms within 6 genes involved in repair and degenerative processes (DEFB1, DENND2C, ESRRB, FGF3, FGF10, and FGFR1) were investigated in 410 patients, 203 with a diagnosis of RCD and 207 presenting with absence of RCD. Exclusion criteria were patients older than 60 years and younger than 45 years with a history of trauma, rheumatoid arthritis, autoimmune syndrome, pregnancy, and use of corticosteroids. Genomic DNA was obtained from saliva samples. Genetic markers were genotyped with TaqMan real-time polymerase chain reaction. The χ(2) test compared genotypes and haplotype differences between groups. Multivariate logistic regression analyzed the significance of many covariates and the incidence of RCD. RESULTS: Statistical analysis revealed female sex (P = .001; odds ratio, 2.07 [1.30-3.30]) and being white (P = .002; odds ratio, 1.88 [1.21-2.90]) to be risk factors for RCD development. A significant association of haplotypes CCTTCCAG in ESRRB (P = .05), CGACG in FGF3 (P = .01), CC in DEFB1 (P = .03), and FGFR1 rs13317 (P = .02) with RCD could be observed. Also, association between FGF10 rs11750845 (P = .03) and rs1011814 (P = .01) was observed after adjustment by ethnic group and sex. CONCLUSIONS: Our work clearly supports the role of DEFB1, ESRRB, FGF3, FGF10, and FGFR1 genes in RCD. Identification of these variants can clarify causal pathways and provide a clue for therapeutic targets.

Polymorphisms in BMP4 and FGFR1 genes are associated with fracture non-union.

Fracture healing is a complex process influenced by a multitude of factors and expression of several thousand genes. Polymorphisms in these genes can lead to an extended healing process and explain why certain patients are more susceptible to develop non-union. A total of 16 SNPs within five genes involved in bone repair pathogenesis (FAM5C, BMP4, FGF3, FGF10, and FGFR1) were investigated in 167 patients with long bone fractures, 101 with uneventful healing, and 66 presenting aseptic non-unions. Exclusion criteria were patients presenting pathological fractures, osteoporosis, hypertrophic and infected non-unions, pregnancy, and children. All genetic markers were genotyped using TaqMan real-time PCR. Chi-square test was used to compare genotypes, allele frequencies, and haplotype differences between groups. Binary logistic regression analyzed the significance of many covariates and the incidence of non-union. Statistical analysis revealed open fracture to be a risk factor for non-union development (p < 0.001, OR 3.6 [1.70-7.67]). A significant association of haplotype GTAA in BMP4 (p = 0.01) and FGFR1 rs13317 (p = 0.005) with NU could be observed. Also, uneventful healing showed association with FAM5C rs1342913 (p = 0.04). Our work supported the role of BMP4 and FGFR1 in NU fracture independently of the presence of previously described risk factors.

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.

Enamel formation genes influence enamel microhardness before and after cariogenic challenge.

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes may contribute to caries. For the present study, we used DNA samples collected from 1,831 individuals from various population data sets. Single nucleotide polymorphism markers were genotyped in selected genes (ameloblastin, amelogenin, enamelin, tuftelin, and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Associations with caries experience can be detected but they are not necessarily replicated in all population groups and the most expressive results was for a marker in AMELX (p=0.0007). To help interpret these results, we evaluated if enamel microhardness changes under simulated cariogenic challenges are associated with genetic variations in these same genes. After creating an artificial caries lesion, associations could be seen between genetic variation in TUFT1 (p=0.006) and TUIP11 (p=0.0006) with enamel microhardness. Our results suggest that the influence of genetic variation of enamel formation genes may influence the dynamic interactions between the enamel surface and the oral cavity.