Analyses of estrogen receptor alpha genetic polymorphisms in Brazilian women with TMJ internal derangement

Orientadores: Celia Marisa Rizzatti-Barbosa, Sergio Roberto Peres Line, Aarão Mendes Pinto NetoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Disfunções temporomandibulares (DTM) são condições encontradas na clínica que causam disfunção e dor no sistema mastigatório, promovidas por uma combinação de fatores, dentre os quais se incluem os hormonais e genéticos. Mulheres são marcadamente mais acometidas por estas desordens e, ao mesmo tempo, indivíduos submetidos à multifatoriedade etiológica das DTM respondem diferentemente quanto ao desenvolvimento e progressão da doença. Em conjunto, essas informações permitem sugerir uma correlação entre os hormônios sexuais femininos estrogênicos e a fisiopatologia das DTM, além de indicarem que condições genéticas inerentes ao indivíduo possam modular a capacidade patogênica do estrógeno. Os efeitos dos estrógenos no organismo são mediados pela sua ligação a receptores específicos localizados no citosol e núcleo celular, dos quais o receptor a (REa) é o mais conhecido e estudado. Em face disso, esta dissertação teve como objetivo investigar a associação de dois polimorfismos de nucleotídeo único localizados no íntron 1 do gene que codifica o receptor de estrógeno alfa e que são identificados pelas enzimas de restrição PvuII e XbaI, com os sinais e sintomas de desarranjos internos da articulação temporomandibular (ATM) em mulheres brasileiras na pós-menopausa, fazendo ou não terapia por reposição hormonal. Para isso, foi realizada a genotipagem destes polimorfismos para 284 mulheres na pós-menopausa, divididas em seis grupos de acordo com a presença ou ausência de desarranjos internos da articulação temporomandibular - dada pelo critério de diagnóstico em pesquisa para desordens temporomandibulares (RDC/TMD) - e com seu estado hormonal - hipoestrogenia ou sob terapia de reposição hormonal (TRH): Controle (Menopausa); DTM sem dor (Menopausa); DTM com dor (Menopausa); Controle (TRH); DTM sem dor (TRH); e DTM com dor (TRH). A ausência dos sítios de restrição para das enzimas PvuII e XbaI foi convencionalmente indicada com as letras maiúsculas P e X, e sua presença com as letras minúsculas p e x, respectivamente. Os sujeitos, portanto, foram designados como homozigotos PP ou XX, pp ou xx, ou ainda, heterozigotos Pp ou Xx. A comparação da freqüência dos genótipos entre os grupos controles e cada um dos grupos de pacientes portadores de desarranjos internos de DTM foi realizada através do teste ?2, com x nível de significância em 5%. O risco associado aos alelos foi calculado, quando pertinente, através do teste Odds Ratio (OR) com 95% de intervalo de confiança (CI). Como resultados, o genótipo homozigoto PP no grupo DTM sem dor (TRH) representou, em relação ao heterozigoto Pp, um risco 0,3125 vezes maior para DTM sem dor quando comparado com o grupo Controle (Menopausa) e um risco 0,2051 vezes maior para a mesma condição quando comparado ao grupo Controle (TRH). Já para o sítio polimórfico reconhecido pela enzima de restrição XbaI, nenhuma associação efetiva pôde ser encontrada. Consideradas as limitações deste estudo, os resultados encontrados permitiram sugerir que o genótipo homozigoto PP no íntron 1 do REa pode, na presença de estrógeno, ser considerado um marcador de risco para o desenvolvimento de patologias articulares na ATM de mulheres brasileiras.Abstract: Disfunções temporomandibulares (DTM) são condições encontradas na clínica que causam disfunção e dor no sistema mastigatório, promovidas por uma combinação de fatores, dentre os quais se incluem os hormonais e genéticos. Mulheres são marcadamente mais acometidas por estas desordens e, ao mesmo tempo, indivíduos submetidos à multifatoriedade etiológica das DTM respondem diferentemente quanto ao desenvolvimento e progressão da doença. Em conjunto, essas informações permitem sugerir uma correlação entre os hormônios sexuais femininos estrogênicos e a fisiopatologia das DTM, além de indicarem que condições genéticas inerentes ao indivíduo possam modular a capacidade patogênica do estrógeno. Os efeitos dos estrógenos no organismo são mediados pela sua ligação a receptores específicos localizados no citosol e núcleo celular, dos quais o receptor a (REa) é o mais conhecido e estudado. Em face disso, esta dissertação teve como objetivo investigar a associação de dois polimorfismos de nucleotídeo único localizados no íntron 1 do gene que codifica o receptor de estrógeno alfa e que são identificados pelas enzimas de restrição PvuII e XbaI, com os sinais e sintomas de desarranjos internos da articulação temporomandibular (ATM) em mulheres brasileiras na pós-menopausa, fazendo ou não terapia por reposição hormonal. Para isso, foi realizada a genotipagem destes polimorfismos para 284 mulheres na pós-menopausa, divididas em seis grupos de acordo com a presença ou ausência de desarranjos internos da articulação temporomandibular - dada pelo critério de diagnóstico em pesquisa para desordens temporomandibulares (RDC/TMD) - e com seu estado hormonal - hipoestrogenia ou sob terapia de reposição hormonal (TRH): Controle (Menopausa); DTM sem dor (Menopausa); DTM com dor (Menopausa); Controle (TRH); DTM sem dor (TRH); e DTM com dor (TRH). A ausência dos sítios de restrição para das enzimas PvuII e XbaI foi convencionalmente indicada com as letras maiúsculas P e X, e sua presença com as letras minúsculas p e x, respectivamente. Os sujeitos, portanto, foram designados como homozigotos PP ou XX, pp ou xx, ou ainda, heterozigotos Pp ou Xx. A comparação da freqüência dos genótipos entre os grupos controles e cada um dos grupos de pacientes portadores de desarranjos internos de DTM foi realizada através do teste ?2, com x nível de significância em 5%. O risco associado aos alelos foi calculado, quando pertinente, através do teste Odds Ratio (OR) com 95% de intervalo de confiança (CI). Como resultados, o genótipo homozigoto PP no grupo DTM sem dor (TRH) representou, em relação ao heterozigoto Pp, um risco 0,3125 vezes maior para DTM sem dor quando comparado com o grupo Controle (Menopausa) e um risco 0,2051 vezes maior para a mesma condição quando comparado ao grupo Controle (TRH). Já para o sítio polimórfico reconhecido pela enzima de restrição XbaI, nenhuma associação efetiva pôde ser encontrada. Consideradas as limitações deste estudo, os resultados encontrados permitiram sugerir que o genótipo homozigoto PP no íntron 1 do REa pode, na presença de estrógeno, ser considerado um marcador de risco para o desenvolvimento de patologias articulares na ATM de mulheres brasileiras.MestradoProtese DentalMestre em Clínica Odontológic

Classes sociais e a educação do campo: resultados preliminares de estudo realizado no Município de Chopinzinho-PR

Anais do II Seminário Seminário Estadual PIBID do Paraná: tecendo saberes / organizado por Dulcyene Maria Ribeiro e Catarina Costa Fernandes — Foz do Iguaçu: Unioeste; Unila, 2014Compreender a realidade dos educandos a partir do conceito de classe tem sido um desafio para a Escola Estadual do Campo Santa Inês, principalmente dentro da proposta de Educação do Campo, que tem se proposto a implementar. Identificar e reconhecer a diversidade e origem histórica dos educandos pode levar a uma perspectiva multicultural, que pode não passar apenas do reconhecimento das diversidades, ignorando a classe a que pertencemos. No entanto, não há como identificar as classes que os educandos pertencem, sem que haja uma aproximação com a realidade dos sujeitos do campo, o que se buscou através do Pibid na escol

Painful Temporomandibular Disorder: Decade of Discovery from OPPERA Studies

In 2006, the OPPERA project (Orofacial Pain: Prospective Evaluation and Risk Assessment) set out to identify risk factors for development of painful temporomandibular disorder (TMD). A decade later, this review summarizes its key findings. At 4 US study sites, OPPERA recruited and examined 3,258 community-based TMD-free adults assessing genetic and phenotypic measures of biological, psychosocial, clinical, and health status characteristics. During follow-up, 4% of participants per annum developed clinically verified TMD, although that was a “symptom iceberg” when compared with the 19% annual rate of facial pain symptoms. The most influential predictors of clinical TMD were simple checklists of comorbid health conditions and nonpainful orofacial symptoms. Self-reports of jaw parafunction were markedly stronger predictors than corresponding examiner assessments. The strongest psychosocial predictor was frequency of somatic symptoms, although not somatic reactivity. Pressure pain thresholds measured at cranial sites only weakly predicted incident TMD yet were strongly associated with chronic TMD, cross-sectionally, in OPPERA’s separate case-control study. The puzzle was resolved in OPPERA’s nested case-control study where repeated measures of pressure pain thresholds revealed fluctuation that coincided with TMD’s onset, persistence, and recovery but did not predict its incidence. The nested case-control study likewise furnished novel evidence that deteriorating sleep quality predicted TMD incidence. Three hundred genes were investigated, implicating 6 single-nucleotide polymorphisms (SNPs) as risk factors for chronic TMD, while another 6 SNPs were associated with intermediate phenotypes for TMD. One study identified a serotonergic pathway in which multiple SNPs influenced risk of chronic TMD. Two other studies investigating gene-environment interactions found that effects of stress on pain were modified by variation in the gene encoding catechol O-methyltransferase. Lessons learned from OPPERA have verified some implicated risk factors for TMD and refuted others, redirecting our thinking. Now it is time to apply those lessons to studies investigating treatment and prevention of TMD

Preclinical orofacial pain assays and measures and chronic primary orofacial pain research: where we are and where we need to go

Chronic primary orofacial pain (OFP) conditions such as painful temporomandibular disorders (pTMDs; i.e., myofascial pain and arthralgia), idiopathic trigeminal neuralgia (TN), and burning mouth syndrome (BMS) are seemingly idiopathic, but evidence support complex and multifactorial etiology and pathophysiology. Important fragments of this complex array of factors have been identified over the years largely with the help of preclinical studies. However, findings have yet to translate into better pain care for chronic OFP patients. The need to develop preclinical assays that better simulate the etiology, pathophysiology, and clinical symptoms of OFP patients and to assess OFP measures consistent with their clinical symptoms is a challenge that needs to be overcome to support this translation process. In this review, we describe rodent assays and OFP pain measures that can be used in support of chronic primary OFP research, in specific pTMDs, TN, and BMS. We discuss their suitability and limitations considering the current knowledge of the etiology and pathophysiology of these conditions and suggest possible future directions. Our goal is to foster the development of innovative animal models with greater translatability and potential to lead to better care for patients living with chronic primary OFP

GWAS Identifies New Loci for Painful Temporomandibular Disorder: Hispanic Community Health Study/Study of Latinos

Temporomandibular disorder (TMD) is a musculoskeletal condition characterized by pain and reduced function in the temporomandibular joint and/or associated masticatory musculature. Prevalence in the United States is 5% and twice as high among women as men. We conducted a discovery genome-wide association study (GWAS) of TMD in 10,153 participants (769 cases, 9,384 controls) of the US Hispanic Community Health Study/Study of Latinos (HCHS/SOL). The most promising single-nucleotide polymorphisms (SNPs) were tested in meta-analysis of 4 independent cohorts. One replication cohort was from the United States, and the others were from Germany, Finland, and Brazil, totaling 1,911 TMD cases and 6,903 controls. A locus near the sarcoglycan alpha (SGCA), rs4794106, was suggestive in the discovery analysis (P = 2.6 × 106) and replicated (i.e., 1-tailed P = 0.016) in the Brazilian cohort. In the discovery cohort, sex-stratified analysis identified 2 additional genome-wide significant loci in females. One lying upstream of the relaxin/insulin-like family peptide receptor 2 (RXP2) (chromosome 13, rs60249166, odds ratio [OR] = 0.65, P = 3.6 × 10−8) was replicated among females in the meta-analysis (1-tailed P = 0.052). The other (chromosome 17, rs1531554, OR = 0.68, P = 2.9 × 10−8) was replicated among females (1-tailed P = 0.002), as well as replicated in meta-analysis of both sexes (1-tailed P = 0.021). A novel locus at genome-wide level of significance (rs73460075, OR = 0.56, P = 3.8 × 10−8) in the intron of the dystrophin gene DMD (X chromosome), and a suggestive locus on chromosome 7 (rs73271865, P = 2.9 × 10−7) upstream of the Sp4 Transcription Factor (SP4) gene were identified in the discovery cohort, but neither of these was replicated. The SGCA gene encodes SGCA, which is involved in the cellular structure of muscle fibers and, along with DMD, forms part of the dystrophin-glycoprotein complex. Functional annotation suggested that several of these variants reside in loci that regulate processes relevant to TMD pathobiologic processes

Modification of COMT-dependent pain sensitivity by psychological stress and sex

Catecholamine-O-methyltransferase (COMT) is a polymorphic gene whose variants affect enzymatic activity and pain sensitivity via adrenergic pathways. Although COMT represents one of the most studied genes in human pain genetics, findings regarding its association with pain phenotypes are not always replicated. Here, we investigated if interactions among functional COMT haplotypes, stress, and sex can modify the effect of COMT genetic variants on pain sensitivity. We tested these interactions in a cross-sectional study, including 2 cohorts, one of 2972 subjects tested for thermal pain sensitivity (Orofacial Pain: Prospective Evaluation and Risk Assessment) and one of 948 subjects with clinical acute pain after motor vehicle collision (post-motor vehicle collision). In both cohorts, the COMT high-pain sensitivity (HPS) haplotype showed robust interaction with stress and number of copies of the HPS haplotype was positively associated with pain sensitivity in nonstressed individuals, but not in stressed individuals. In the post-motor vehicle collision cohort, there was additional modification by sex: the HPS-stress interaction was apparent in males, but not in females. In summary, our findings indicate that stress and sex should be evaluated in association studies aiming to investigate the effect of COMT genetic variants on pain sensitivity

Molecular genetic mechanisms of allelic specific regulation of murine Comt expression

A functional allele of the mouse catechol-O-methyltransferase (Comt) gene is defined by the insertion of a B2 short interspersed repeat element in its 3′-untranslated region (UTR). This allele has been associated with a number of phenotypes, such as pain and anxiety. In comparison with mice carrying the ancestral allele (Comt+), ComtB2i mice show higher Comt mRNA and enzymatic activity levels. Here, we investigated the molecular genetic mechanisms underlying this allelic specific regulation of Comt expression. Insertion of the B2 element introduces an early polyadenylation signal generating a shorter Comt transcript, in addition to the longer ancestral mRNA. Comparative analysis and in silico prediction of Comt mRNA potential targets within the transcript 3′ to the B2 element was performed and allowed choosing microRNA (miRNA) candidates for experimental screening: mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667. Cell transfection with each miRNA downregulated the expression of the ancestral transcript and COMT enzymatic activity. Our in vivo experiments showed that mmu-miR-667-3p is strongly correlated with decreasing amounts of Comt mRNA in the brain, and lentiviral injections of mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667 increase hypersensitivity in the mouse formalin model, consistent with reduced COMT activity. In summary, our data demonstrate that the Comt+ transcript contains regulatory miRNA signals in its 3′-untranslated region leading to mRNA degradation; these signals, however, are absent in the shorter transcript, resulting in higher mRNA expression and activity levels

COMT gene locus: new functional variants

Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3′ untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes

The genetics of neuropathic pain from model organisms to clinical application

Neuropathic pain (NeuP) arises due to injury of the somatosensory nervous system and is both common and disabling, rendering an urgent need for non-addictive, effective new therapies. Given the high evolutionary conservation of pain, investigative approaches from Drosophila mutagenesis to human Mendelian genetics have aided our understanding of the maladaptive plasticity underlying NeuP. Successes include the identification of ion channel variants causing hyper-excitability and the importance of neuro-immune signaling. Recent developments encompass improved sensory phenotyping in animal models and patients, brain imaging, and electrophysiology-based pain biomarkers, the collection of large well-phenotyped population cohorts, neurons derived from patient stem cells, and high-precision CRISPR generated genetic editing. We will discuss how to harness these resources to understand the pathophysiological drivers of NeuP, define its relationship with comorbidities such as anxiety, depression, and sleep disorders, and explore how to apply these findings to the prediction, diagnosis, and treatment of NeuP in the clinic

Epiregulin and EGFR interactions are involved in pain processing

The EGFR belongs to the well-studied ErbB family of receptor tyrosine kinases. EGFR is activated by numerous endogenous ligands that promote cellular growth, proliferation, and tissue regeneration. In the present study, we have demonstrated a role for EGFR and its natural ligand, epiregulin (EREG), in pain processing. We show that inhibition of EGFR with clinically available compounds strongly reduced nocifensive behavior in mouse models of inflammatory and chronic pain. EREG-mediated activation of EGFR enhanced nociception through a mechanism involving the PI3K/AKT/mTOR pathway and matrix metalloproteinase-9. Moreover, EREG application potentiated capsaicin-induced calcium influx in a subset of sensory neurons. Both the EGFR and EREG genes displayed a genetic association with the development of chronic pain in several clinical cohorts of temporomandibular disorder. Thus, EGFR and EREG may be suitable therapeutic targets for persistent pain conditions