Polimorfismos de nucleotídeo único (SNPS) dos genes PPARy2, lipase lipoproteica, receptor de LDL, apolipoproteina C3, e adiponectina podem modular o perfil lipídico de pacientes com a síndrome de Berardinelli-Seip

A Síndrome de Berardinelli-Seip (SBS) é um distúrbio raro do metabolismo dos lipídios, caracterizada pela ausência quase total de tecido adiposo subcutâneo, hipertrigliceridemia, hipoleptinemia e diabetes insulino resistente ou lipoatrófico. Sua etiologia envolve implicações hipotalâmicas, alterações nos receptores de insulina e mutações nos genes AGPAT2, Gng3lg, CAV1 e PTRF. O tecido adiposo secreta diversas substâncias, tais como: leptina, resistina, adiponectina, esteróides, TNF , IL-6, PAI-1, angiotensinogênio, IGF-1. Muitas delas estão associadas ao diabetes mellitus tipo 2, obesidade e hipertensão. Os PPARs são fatores transcricionais pertencentes à superfamília de receptores nucleares ligantes ativados. Sabe-se que o PPAR , é importante para o metabolismo lipídico e glicídico e que o ligante natural do PPAR é derivado do ácido graxo. Nesse sentido, foram avaliados 24 pacientes portadores da SBS, provenientes do Estado do Rio Grande do Norte, com a mediana das idades de 18,5 anos (0,55 a 47 a), sendo 9 (37,5 %) do gênero masculino e 15 (62,5 %) do gênero feminino. Quanto ao grupo étnico, foram classificados em caucasóides (brancos) 21 (87,5 %) e negróides 3 (12,5 %) pacientes. Foram feitas avaliações clínico-endocrinológica, bioquímica, hormonal, molecular e o estudo dos polimorfismos Adiponectina ADIPOQ, PPARγ2 Pro12Ala, LPL-PvuII, APOC3-SstI e LDLR-AvaII em portadores da SBS. Nesta população nós não encontramos nenhuma associação de parâmetros lipídicos e glicídicos com os polimorfismos LPL-PvuII, APOC3-SstI e LDLR-AvaII. Porém, observamos associação entre Adiponectina ADIPOQ e PPARγ2 Pro12Ala e níveis lipídicos mais elevados, sugerindo um papel biológico para estes fatores, indicando estudos mais aprofundados

Genes candidatos de suscetibilidade a pré-eclampsia: estudo de associação

Preeclampsia is a multifactorial disease of unknown etiology that features with wide clinical symptoms, ranging from mild preeclampsia to severe forms, as eclampsia and HELLP syndrome. As a complex disease, preeclampsia is also influenced by genetic and environmental factors. Aiming to identify preeclampsia susceptibility genes, we genotyped a total of 22 genetic markers (single nucleotides polymorphisms SNPs) distributed in six candidates genes (ACVR2A, FLT1, ERAP1, ERAP2, LNPEP e CRHBP). By a case-control approach, the genotypic frequencies were compared between normotensive (control group) and preeclamptic women. The case s group was classified according to the disease clinical form in: preeclampsia, eclampsia and HELLP syndrome. As results we found the following genetic association: 1) ACVR2A and preeclampsia; 2) FLT1 and severe preeclampsia; 3) ERAP1 and eclampsia; 4) FLT1 and HELLP syndrome. When stratifying preeclampsia group according to symptoms severity (mild and severe preeclampsia) or according to the time of onset (early and late preeclampsia), it was detected that early preeclampsia is strongly associated to risk preeclampsia, eclampsia and HELLP syndrome have different genetic bases, although FLT1 gene seems to be involved in preeclampsia and HELLP syndrome pathophisiology

Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Análise da Expressão de APE1 após Estresse Oxidativo Induzido em Células Proficientes e Deficientes na Via de Reparo por Excisão de Nucleotídeos

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.

Marcadores genéticos associados com a resposta inflamatória na meningite bacteriana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Análise de polimorfismos dos genes da rota quinureniana em pacientes com meningite bacteriana.

Bacterial meningitis (BM) is still an important infectious disease causing death and disability. Invasive bacterial infections of the central nervous systems (CNS) generate some of the most powerful inflammatory responses known, which contributes to neuronal damage. The DNA microarray technology showed alterations in the kynurenine (KYN) pathway that is induced in BM and other diseases associated with inflammation, leading to brain injury. Our main aim was to search SNPs previously described in the KYN path enzymes to investigate a putative association of this SNPs with imbalanced in this pathway in patients with BM. The patients included in this study were 33 males and 24 females, with ages varying from 02 months to 68 years. SNPs were located inside of the domain conserved in KYNU, IDO, KATI and KATII. Primers were designed for analysis of SNPs already described by PIRA-PCR followed by RFLP. The analysis of KYNU+715G/A SNP found a heterozygous frequency of 0.033. We did not found the variant allele of SNP KYNU+693G/A, KATI+164T/C, KATII+650C/T and IDO+434T/G. Despite of previews studies showing the importance of KYN pathway we did not found one association of these SNPs analyzed with susceptibility or severity of MB in study population.

Associação entre polimorfismos em enzimas de reparo de DNA e ocorrência de meningite bacteriana.

Despite advances in vaccine development and therapy, bacterial meningitis (BM) remains a major cause of death and long-term neurological disabilities. As part of the host inflammatory response to the invading pathogen, factors such as reactive oxygen species are generated, which may damage DNA and trigger the overactivation of DNA repair mechanisms. It is conceivable that the individual susceptibility and outcome of BM may be in part determined by non synonymous polymorphisms that may alter the function of crucial BER DNA repair enzymes as PARP-1, OGG-1 and APE-1. These enzymes, in addition to their important DNA repair function, also perform role of inflammatory regulators. In this work was investigated the non synonymous SNPs APE-1 Asn148Glu, OGG-1 Ser326Cys,PARP-1 Val762Ala, PARP-1 Pro882Leu and PARP-1 Cys908Tyr in patients with bacterial meningitis (BM), chronic meningitis (CM), aseptic meningitis (AM) and not infected (controls). As results we found increased frequency of variant alleles of PARP-1 Val762Ala (P = 0.005) and APE-1 Asn148Glu (P=0.018) in BM patients, APE-1 Asn148Glu in AM patients (P = 0.012) and decrease in the frequency of the variant allele OGG-1 Ser326Cys in patients with CM (P = 0.013), regarding the allelic frequencies in the controls. A major incidence of individuals heterozygous and/ or polymorphic homozygous in BM for PARP-1 Val762Ala (P= 0.0399, OD 4.2, 95% IC 1.213 -14.545) and PARP-1 Val762Ala/ APE-1 Asn148Glu (P = 0.0238, OD 11.111, 95% IC 1.274 - 96.914) was observed related to what was expected in a not infected population. It was also observed a major incidence of combined SNPs in the BM patients compared with the control group (P=0.0281), giving evidences that SNPs can cause some susceptibility to the disease. This combined effect of SNPs seems to regulate the principal cytokines and other factors related to BM inflammatory response and point the importance of DNA repair not only to repair activity when DNA is damaged, but to others essential functions to human organism balance.

Associação entre polimorfismos funcionais nos genes da MMP-7 e MMP-9 e o perfil clinicopatológico do carcinoma epidermóide de língua

Matrix metalloproteinase-7 (MMP-7) and -9 (MMP-9) modulate important functions strictly related to the development, invasion and metastasis of several human cancers among them the squamous cell carcinoma of the tongue (SCCT). However, individual genetic factors such as the functional single nucleotide polymorphisms (SNPs) influence the pattern of protein expression of these MMPs and thus may be related to the variability observed in the clinical behavior of patients with SCCT. In this context, the present cross-sectional study aimed to evaluate the association between the frequency of the functional SNPs MMP-7 -181 A/G and MMP-9 -1562 C/T and the clinical (age, gender and metastasis) and pathological (malignancy histological grading and immunohistochemistry expression) features of SCCT cases. Genotyping of these SNPs were performed by PCR-RFLP on DNA samples from 71 cases of SCCT and 60 individuals without cancer who constitute the control group. Among the results of this research, it was observed that the frequency of the polymorphic alleles MMP-7 -181 G and MMP-9 -1562 T in SCCT patients was 28% and 12%, respectively, and the frequency of the heterozygotes A/G (PR = 2.00; p < 0.001) and C/T (PR = 1.54; p = 0.014) were significantly higher in the patient group than in the controls. The prevalence of patients carrying the combination of SNPs studied was significantly associated with SCCT cases (PR = 2.00; p = 0.011) and metastasis (PR = 2.00; p < 0.001). Furthermore, with the frequency of SNPs analyzed, the age, gender, histological grading and immunoreactivity of MMP-7 and MMP-9 formed clinical and pathological parameters relevant to the identification of population subgroups more related to the development of SCCT and metastasis. Based on these results, it is suggested that the protein expression levels of MMP-7 and -9 substantially influence the balance between their pro- and anticancer biological functions and hence the clinicopathological profile of the squamous cell carcinoma of the tongue