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Several genes have been associated with breast cancer (BC) susceptibility. The tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A), and interferon lambda receptor 1 (IFNLR1) genes encode receptors that mediate the action of inflammatory cytokines. Previous studies have demonstrated the association of the variants rs1800693 (TNFRSF1A) and rs4649203 (IFNLR1) with some inflammatory diseases. The present study aimed to verify a possible association of these variants with BC, its clinical pathologic features, as well as epidemiological data in a Brazilian population. A total of 243 patients and 294 individuals without history of BC were genotyped for these polymorphisms through TaqMan® SNP genotyping assays by qPCR. For the TNFRSF1A gene, no significant results were found. For IFNLR1, the AA genotype (p = 0.008) and the A allele (p = 0.02) were significantly associated with a lower risk of developing BC. When analyzing the age, it was observed that each increase of one year contributes to the development of BC (p

O câncer de mama é o tipo mais comum entre as mulheres. O sistema imune tem um papel essencial no controle de neoplasias, que se desenvolvem através do mecanismo de escape tumoral. Um gene imunorregulador com papel nesse mecanismo é o HLA-G. Uma deleção na região codificadora desse gene gera o alelo nulo G*0105N que impede a expressão de algumas isoformas, podendo estar relacionado a uma menor susceptibilidade ao câncer de mama. O objetivo deste estudo foi analisar a presença do alelo nulo do HLA-G em 106 mulheres com câncer de mama e em 96 mulheres sem evidência da neoplasia, para investigar uma possível associação entre o polimorfismo e a doença. Foi também realizado um levantamento de dados epidemiológicos, que foram testados como fatores de risco. O DNA foi extraído e a genotipagem feita por PCR – RFLP. Não foi observada a presença do polimorfismo no gene HLA-G em nenhuma das amostras analisadas. A prevalência do G*0105Nvaria entre as populações, resultado de processos evolutivos distintos nos diferentes grupos populacionais. Para os dados epidemiológicos a idade média para casos foi de 49 anos; 64% tiveram a menarca entre 12 e 14 anos; 78% amamentaram em algum período da vida; 72% fizeram uso de contraceptivos hormonais por um período de até 60 meses; e o tipo histológico de câncer mais incidente (72%) foi o Ductal Invasivo. Embora o polimorfismo não tenha sido detectado nas amostras analisadas, a análise dos dados epidemiológicos reforça a participação desses fatores no desenvolvimento da doença. Breast cancer (BRCA) is the most common type of cancer among women. Immune system plays an essential role in controlling of neoplasia that are developed through the tumor escaping mechanism. An immunoregulatory gene that plays a main role in this mechanism is HLA-G. A deletion in the coding region of this gene promotes the G * 0105Nwich is a null allele that prevents the expression of some isoforms and may be related to a lower susceptibility to BRCA. In this study was to analyze the presence of the HLA-G null allele in 106 women with BRCA and in 96 women with no evidence of the disease in order to investigate a possible association between this polymorphism and disease. Epidemiological data were tested as a risk factor for a disease. DNA was extracted and genotyping done by PCR-RFLP. Presence of polymorphism in the HLA-G gene was not observed in any of the samples analyzed. Prevalence of G*0105N oscillate among populations, as a result from distinct evolutionary processes in different population groups. For the epidemiological data the average age for cases was 49 years old; 64% – were menarche between 12 and 14 years; 78% breastfed at some point of life; 72% used hormonal contraceptives for up to 60 months; and the most common (72%) histological type of cancer was Ductal Invasive. Although the polymorphism was not detected in the analyzed samples, the analysis of epidemiological data reinforces the participation of these factors in the development of the disease.

HLA-G is a promiscuous immune checkpoint molecule. The HLA-G gene presents substantial nucleotide variability in its regulatory regions. However, it encodes a limited number of proteins compared to classical HLA class I genes. We characterized the HLA-G genetic variability in 4640 individuals from 88 different population samples across the globe by using a state-of-the-art method to characterize polymorphisms and haplotypes from high-coverage next-generation sequencing data. We also provide insights regarding the HLA-G genetic diversity and a resource for future studies evaluating HLA-G polymorphisms in different populations and association studies. Despite the great haplotype variability, we demonstrated that: (1) most of the HLA-G polymorphisms are in introns and regulatory sequences, and these are the sites with evidence of balancing selection, (2) linkage disequilibrium is high throughout the gene, extending up to HLA-A, (3) there are few proteins frequently observed in worldwide populations, with lack of variation in residues associated with major HLA-G biological properties (dimer formation, interaction with leukocyte receptors). These observations corroborate the role of HLA-G as an immune checkpoint molecule rather than as an antigen-presenting molecule. Understanding HLA-G variability across populations is relevant for disease association and functional studies., We acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil, #302060/2019-7), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/Brazil, Finance code: 001), INCTC/FAPESP (Grant #465539/2014-9) for financial support, and FAPESP/Brazil (#Grant 2017/19223-0).

O gene HLA-G apresenta considerável variabilidade nucleotídica nas suas regiões reguladoras, mas limitada variabilidade proteica e restrita expressão tecidual, em condições não-patológicas. A proteína HLA-G modula a atividade de células do sistema imune inato e adquirido e tem sido explorada em várias condições fisiológicas e patológicas, nas quais essas respostas podem ser benéficas ou deletérias, dependendo das características dessas condições. Considerando que a magnitude da expressão de HLA-G pode ser regulada pelas regiões promotora e 3\'NT e que a expressão diferencial de HLA-G foi descrita mesmo em indivíduos saudáveis, neste trabalho avaliamos a diversidade das regiões regulatórias do gene HLA-G e sua possível influência nas variações de expressão da proteína em condições fisiológicas e patológicas. Para isso, realizamos: (i) uma revisão sistemática seguida de meta-análise sobre o polimorfismo mais estudado da região 3\'NT do gene (presença ou ausência de 14-pb) em doenças de diversas etiologias; (ii) a tipificação das regiões regulatórias do HLA-G (sequenciamento de nova geração) em pacientes com doença de Chagas, para verificação de possíveis associações entre os polimorfismos e a doença, e a avaliação dos níveis plasmáticos (ELISA) de HLA-G e da expressão tecidual (em coração, colón e esôfago) da proteína HLA-G (imunoistoquímica); (iii) a análise da diversidade das regiões promotoras (segmentos distal, proximal e enhancer L) e 3\'NT do gene HLA-G nas diversas populações mundiais que compõem a base de dados 1000 Genomes - Fase 3, para identificação dos principais haplótipos, suas frequências e relações com as diferentes regiões geográficas; e (iv) a associação da variabilidade das regiões promotoras (distal e proximal) e 3\'NT do HLA-G com os níveis plasmáticos de sHLA-G em indivíduos saudáveis, para identificar possíveis interferências das variações nucleotídicas dessas regiões na expressão basal da proteína HLA-G. De maneira geral, a meta-análise revelou que a variabilidade dos 14 pares de bases na região 3\'NT não é um marcador genético eficiente para estudos de associação de doenças. Embora os níveis plasmáticos de sHLA-G e a expressão tecidual de HLA-G estivessem mais elevados pacientes 9 com doença de Chagas quando comparados com respectivos controles, não observamos associação entre os sítios polimórficos das regiões promotora e 3\'NT do gene com a doença. Nas diversas populações mundiais, os alelos mais frequentes observados nas regiões reguladoras do gene HLA-G apresentam-se com frequências similares e verificou-se indícios de seleção balanceadora atuando nesses segmentos gênicos. Além disso, foi verificado um alto desequilíbrio de ligação entre os pares de SNP que compreendem toda essa região. Na população brasileira do Nordeste do Estado de São Paulo, não observamos associação entre os níveis de sHLA-G com a variabilidade das regiões reguladoras do gene. Por fim, considerando que a modulação da expressão de HLA-G pode ter possível aplicação clínica, o conhecimento da diversidade das regiões reguladoras pode contribuir para o entendimento acerca da complexidade da regulação da expressão e da magnitude de expressão do gene. HLA-G gene has considerable nucleotide variability in its regulatory regions, but limited protein variability and restricted tissue expression under non-pathological conditions. HLA-G protein modulates the activity of innate and acquired immune system cells and has been exploited under various physiological and pathological conditions, where these responses may be beneficial or deleterious, depending on disease conditions. Considering that the magnitude of HLA-G expression can be regulated by the promoter and 3\' untranslated (3\'UTR) regions and that differential expression of HLA-G was described even in healthy individuals, in this work we evaluated the diversity of the HLA-G gene regulatory regions and its possible influence on protein expression variation under physiological and pathological conditions. For this, we performed: (i) a systematic review followed by meta-analysis of the most studied polymorphism of the HLA-G 3\'UTR (presence or absence of 14-bp) in diseases of several etiologies; (ii) the typing of HLA-G regulatory regions (next generation sequencing) in patients with Chagas disease, to verify possible associations between polymorphisms and disease, and the evaluation of HLA-G plasma levels (ELISA) and tissue expression (in heart, colon and esophagus) of HLA-G protein (immunohistochemistry); (iii) the analysis of the promoter regions (distal, proximal and enhancer segments L) and 3\'UTR diversity on worldwide populations, using the 1000 Genomes - Phase 3 database, to identify the main haplotypes, their frequencies and relations with different geographical regions; and (iv) the association of the variability of HLA-G promoter (distal and proximal) and 3\'UTR segments with sHLA-G plasma levels in healthy individuals, to identify possible interference of nucleotide variations on basal HLA-G protein expression. Overall, the meta-analysis revealed that 14 base pair variability at the 3\'UTR is not an efficient genetic marker for disease association studies. Although sHLA-G plasma levels and HLA-G tissue expression were higher in patients with Chagas disease compared to their controls, we found no association between the polymorphic promoter and 3\'UTR gene regions with disease. In the world population evaluation, the most 11 frequent alleles observed at the regulatory regions of the HLA-G gene are similar in frequency and there was evidence of balancing selection acting on these gene segments. In addition, a high linkage disequilibrium was found between SNP pairs that comprise this entire region. In the Brazilian population of Northeastern São Paulo State, we did not observe any association between sHLA-G levels and the variability of gene regulatory regions. Finally, considering that the modulation of HLA-G expression may have possible clinical application, the knowledge of the diversity of regulatory regions may contribute to the understanding the complexity of HLA-G gene regulation.

Made available in DSpace on 2018-12-11T17:22:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-10-01 Background: HLA-G is an immune checkpoint molecule. Since a differential molecule expression has been reported even for healthy individuals, many studies have focused on polymorphisms at HLA-G regulatory regions, particularly the 3′ untranslated region (3′UTR). The presence/absence of a 14-bp sequence was the first polymorphism described and it is the most studied in association between HLA-G and disorders. Methods: In this study, we performed a systematic review and meta-analysis of all association studies published regarding the HLA-G 14-bp. Results: We verified association between 14-bp alleles and diseases in the following situations: (1) presence of 14-bp (insertion) conferred susceptibility to preeclampsia (child alleles evaluated) and systemic lupus erythematosus (OR = 1.42; 95%CI = 1.04–1.93; p = 0.026 and OR = 1.13; 95%CI = 1.01–1.27, p = 0.028); (2) 14-bp absence (deletion) was associated with increased risk to breast cancer (OR = 1.23; 95%CI = 1.06–1.43; p = 0.006) and human Cytomegalovirus infection (OR = 2.06; 95%CI = 1.60–2.64; p < 0.0001); and (3) a risk association was observed between the group of reproductive disorders and the 14-bp insertion (OR = 1.12; 95%CI = 1.01–1.24; p = 0.034). Conclusions: Considering that others 14-bp associations were inconclusive and that other variation sites observed at HLA-G 3′UTR exhibit a proven role on post-transcriptional regulation of HLA-G expression, the complete 3′UTR segment should be analyzed in terms of disease susceptibility, instead of a single polymorphism. Divisão de Imunologia Clínica Departamento de Clínica Médica Faculdade de Medicina de Ribeirão Preto (FMRP) Universidade de São Paulo (USP) Laboratório Multiusuário de Estudos em Biologia Centro de Ciências Biológicas Universidade Federal de Santa Catarina (UFSC) Departamento de Biologia Celular Embriologia e Genética Centro de Ciências Biológicas Universidade Federal de Santa Catarina (UFSC) Departamento de Patologia Faculdade de Medicina de Botucatu Unesp – Univ. Estadual Paulista Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP) Universidade de São Paulo (USP) Departamento de Patologia Faculdade de Medicina de Botucatu Unesp – Univ. Estadual Paulista

// Layale Yaghi 1, 2, 3 , Isabelle Poras 1, 2 , Renata T. Simoes 1, 2, 4 , Eduardo A. Donadi 5 , Jorg Tost 6, 7 , Antoine Daunay 6 , Bibiana Sgorla de Almeida 1, 2, 5 , Edgardo D. Carosella 1, 2 , Philippe Moreau 1, 2 1 Commissariat a l’Energie Atomique et aux Energies Alternatives, Institut des Maladies Emergentes et des Therapies Innovantes, Service de Recherches en Hemato-Immunologie, Hopital Saint-Louis, Paris, France 2 Universite Paris-Diderot, Sorbonne Paris-Cite, UMR E5, Institut Universitaire d’Hematologie, Hopital Saint-Louis, Paris, France 3 Lebanese University, School of Medicine, Hadath, Lebanon 4 Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, IEP/SCBH, Belo Horizonte, Minas Gerais, Brasil 5 Divisao de Imunologia Clinica, Departamento de Clinica Medica, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo, Brasil 6 Centre d’Etude du Polymorphisme Humain, Fondation Jean-Dausset, Laboratory for Functional Genomics, Paris, France 7 Commissariat a l’Energie Atomique et aux Energies Alternatives, Centre National de Genotypage, Laboratory for Epigenetics and Environment, Evry, France Correspondence to: Philippe Moreau, email: philippe.moreau@cea.fr Keywords: HLA-G, HIF-1, exon 2 HRE, glioma Received: July 02, 2016 Accepted: August 20, 2016 Published: August 26, 2016 ABSTRACT HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G . We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2’deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G . Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at –966 bp in the 5’UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus.

The application of DNA technology in forensic investigations has grown rapidly in the last 25 years and with an exponential increase of short tandem repeats (STRs) data, usually presented as allele frequencies, that may be later used as databases for forensic and population genetics purposes. Thereby, classes of molecular markers such as single nucleotide polymorphisms and insertions/deletions (InDels) have been presented as another option of genetic marker sets. These markers can be used in paternity cases, when mutations in STR polymorphisms are present, as well as in highly degraded DNA analysis. In the present study, the allele frequencies and heterozygosity (H) of a 30 InDel markers set were determined and the forensic efficacy was evaluated through estimation of discrimination power (DP), match probability, typical paternity index and power of paternity exclusion in 108 unrelated volunteers from the State of Santa Catarina (South Brazil). The observed H per locus showed a range between 0.370 and 0.574 (mean = 0.479). HLD128 was the locus with the highest DP (DP = 0.656). DP for all markers combined was greater than 99.9999999999646 % which provides satisfactory levels of information for forensic demands. Genetic comparisons (exact tests of population differentiation and pairwise genetic distances) revealed that the population of Santa Catarina State differs from Korea and USA Afro-American populations but is similar to the Portuguese, German, Polish, Spanish and Basque populations.

Made available in DSpace on 2018-12-11T17:23:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The immune checkpoint HLA-G prevents maternal rejection of the fetus and contributes in cancer invasion and acceptance of allografts. The 5′ and 3′ regulatory regions of the HLA-G gene are polymorphic and balancing selection probably maintains this variability. It is proposed that nucleotide variations may affect the level of HLA-G expression. To investigate this issue we aimed to analyze how haplotypes of the 3′ untranslated region (3′UTR) with highest worldwide frequencies, namely UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18 and UTR-7, impact the expression of a luciferase reporter gene in vitro. Experiments performed with the HLA-G positive cell lines JEG-3 (choricarcinoma) and FON (melanoma), and with the HLA-G negative cell lines M8 (melanoma) and U251MG (glioblastoma) showed that the HLA-G 3′UTR polymorphism influences the response to endogenous cellular factors and may vary according to the cell type. UTR-5 and UTR-7 impact the activity of luciferase the most whereas UTR-2, UTR-3, UTR-4, and UTR-18 have intermediate impact, and UTR-1 has the lowest impact. These results corroborate the previous associations between amounts of plasma sHLA-G levels and 3′UTR haplotypes in healthy individuals and reinforce that 3′UTR typing may be a predictor of the genetic predisposition of an individual to express different levels of HLA-G. Commissariat à l'Energie Atomique et aux Energies Alternatives Institut des Maladies Emergentes et des Thérapies Innovantes Service de Recherches en Hémato-Immunologie Hôpital Saint-Louis Université Paris-Diderot Sorbonne Paris-Cité UMR-E5 Institut Universitaire D'Hématologie Hôpital Saint-Louis Lebanese University School of Medicine Programa de Pósgraduação em Ciências da Saúde Centro de Ciências da Saúde Universidade Federal do Rio Grande do Norte Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Departamento de Biologia Celular Embriologia e Genética Centro de Ciências Biológicas Universidade Federal de Santa Catarina Department of Genetics Ribeirão Preto Medical School University of São Paulo Divisão de Imunologia Clínica Departamento de Clínica Médica Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Department of Pathology School of Medicine Universidade Estadual Paulista Unesp Department of Pathology School of Medicine Universidade Estadual Paulista Unesp CAPES: 014747/2013-8 FAPESP: 2014/18730-9 CNPq: 406594/2013-9

Moderate physical activity when performed on a regular basis presents a number of benefits to the whole organism, especially regarding immune system function, such as augmenting resistance to infections and to cancer growth. Although glutamine production by active muscle cells as well as neuroendocrine alterations mediated by the chronic adaptation to exercise may play a role, the entire mechanism by which exercise makes the immune system aware of challenges remains mostly uncovered. This is particularly true for the effects of an acute exercise session on immune function. In this work, circulating monocytes/macrophages from sedentary rats submitted to an acute (1 h) swimming session were tested for the ability of phagocytosing zymosan particles, phorbol myristate acetate (PMA)-induced hydrogen peroxide production, nitric oxide (NO) release (assessed by nitrate and nitrite production) and the expression of NO synthases (NOS-1, NOS-2 and NOS-3). The results showed that an exercise bout induced a 2.4-fold rise in macrophage phagocytic capacity (p = 0.0041), a 9.6-fold elevation in PMA-induced hydrogen peroxide release into the incubation media (1-h, p = 0.0022) and a 95.5%-augmentation in nitrite basal production (1-h incubation; p = 0.0220), which was associated with a marked expression of NOS-2 (the inducible NOS isoform; p = 0.0319), but not in other NOS gene products. Although NOS-2 expression is nuclear factor-kappaB (NF-kappaB)-dependent, no systemic oxidative stress was found, as inferred from the data of plasma TBARS and glutathione disulphide (GSSG) to glutathione (GSH) ratio in circulating blood erythrocytes which remained constant after the acute exercise. Also, no stressful situation seemed to be faced by monocytes/macrophages, since the expression of the 70-kDa heat shock protein (HSP70) remained unchanged. We conclude that NF-kappaB-dependent induction of NOS-2 and macrophage activation must be related to local factor(s) produced in the surroundings of monocytes/macrophages.

Striated muscle activity is always accompanied by oxidative stress (OxStress): the more intense muscle work and/or its duration, the more a redox imbalance may be attained. In spite of cardiac muscle functioning continuously, it is well known that the heart does not suffer from OxStress-induced damage over a broad physiological range. Although the expression of antioxidant enzymes may be of importance in defending heart muscle against OxStress, a series of combined antioxidant therapeutic approaches have proved to be mostly ineffective in avoiding cellular injury. Hence, additional mechanisms may be involved in heart cytoprotection other than antioxidant enzyme activities. The strong cardiotoxic effect of doxorubicin-induced cancer chemotherapy shed light on the possible role for multidrug resistance-associated proteins (MRP) in this context. Muscle activity-induced ‘physiological’ OxStress enhances the production of glutathione disulfide (GSSG) thus increasing the ratio of GSSG to glutathione (GSH) content inside the cells, which, in turn, leads to redox imbalance. Since MRP1 gene product (a GS-X pump ATPase) is a physiological GSSG transporter, adult Wistar rats were tested for MRP1 expression and activity in the heart and skeletal muscle (gastrocnemius), in as much as the latter is known to be extremely sensitive to muscle activity-induced OxS. MRP1 expression was completely absent in skeletal muscle. In contrast, the heart showed an exercise training-dependent induction of MRP1 protein expression which was further augmented (2.4-fold) as trained rats were challenged with a session of acute exercise. On the other hand, inducible expression of the 70-kDa heat shock protein (HSP70), a universal marker of cellular stress, was completely absent in the heart of sedentary and acutely exercised rats, whereas skeletal muscle showed a conspicuous exercise-dependent HSP70 expression, which decreased by 45% with exercise training. This effect was paralleled by a 58% decrease in GSH content in skeletal muscle which was even higher (an 80%-fall) after training thus leading to a marked redox imbalance ([GSSG]/[GSH] raised up to 38-fold). In the heart, GSH contents and [GSSG]/[GSH] ratio remained virtually unchanged even after exercise challenges, while GS-X pump activity was found to be 20% higher in the heart related to skeletal muscle. These findings suggest that an intrinsic higher capacity to express the MRP1/GS-X pump may dictate the redox status in the heart muscle thus protecting myocardium by preventing GSSG accumulation in cardiomyocytes as compared to skeletal muscle fibres. Copyright © 2006 John Wiley & Sons, Ltd.