RESUMO
BACKGROUND: Rheumatoid arthritis has a complex mode of inheritance. Although HLA-DRB1 and PTPN22 are well-established susceptibility loci, other genes that confer a modest level of risk have been identified recently. We carried out a genomewide association analysis to identify additional genetic loci associated with an increased risk of rheumatoid arthritis. METHODS: We genotyped 317,503 single-nucleotide polymorphisms (SNPs) in a combined case-control study of 1522 case subjects with rheumatoid arthritis and 1850 matched control subjects. The patients were seropositive for autoantibodies against cyclic citrullinated peptide (CCP). We obtained samples from two data sets, the North American Rheumatoid Arthritis Consortium (NARAC) and the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA). Results from NARAC and EIRA for 297,086 SNPs that passed quality-control filters were combined with the use of Cochran-Mantel-Haenszel stratified analysis. SNPs showing a significant association with disease (P<1x10(-8)) were genotyped in an independent set of case subjects with anti-CCP-positive rheumatoid arthritis (485 from NARAC and 512 from EIRA) and in control subjects (1282 from NARAC and 495 from EIRA). RESULTS: We observed associations between disease and variants in the major-histocompatibility-complex locus, in PTPN22, and in a SNP (rs3761847) on chromosome 9 for all samples tested, the latter with an odds ratio of 1.32 (95% confidence interval, 1.23 to 1.42; P=4x10(-14)). The SNP is in linkage disequilibrium with two genes relevant to chronic inflammation: TRAF1 (encoding tumor necrosis factor receptor-associated factor 1) and C5 (encoding complement component 5). CONCLUSIONS: A common genetic variant at the TRAF1-C5 locus on chromosome 9 is associated with an increased risk of anti-CCP-positive rheumatoid arthritis.
Assuntos
Artrite Reumatoide/genética , Cromossomos Humanos Par 9/genética , Complemento C5/genética , Predisposição Genética para Doença , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Fator 1 Associado a Receptor de TNF/genética , Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Estudos de Casos e Controles , Mapeamento Cromossômico , Genótipo , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Humanos , Modelos Logísticos , Peptídeos Cíclicos/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22 , Proteínas Tirosina Fosfatases/genética , Fatores de Risco , Análise de Sequência de DNARESUMO
Previous studies have suggested that the ancestral haplotype 18.2 (AH18.2) carries additional susceptibility gene to Type 1 Diabetes (T1D) on the Major Histocompatibility Complex (MHC). We analyzed 10 DR3/TNFa1b5 homozygous subjects in order to establish the conservation of the AH18.2 and then compared this conserved region with other DR3 haplotype, the AH8.1. The Illumina's HumanHap550 Bead chip was used to perform an extensive genotyping of the MHC region. The AH18.2 was highly conserved between DDR1 and HLA-DQA1 genes; therefore most probably the second susceptibility gene is located within this region. We can exclude the region centromeric to HLA-DRA gene and telomeric to DDR1 gene. A comparison between the AH18.2 and AH8.1 haplotypes showed that 233 SNPs were different in the aforementioned conserved region. These data suggest that the 1.65 Mb MHC region between DDR1 and HLA-DRA genes is likely to carry additional susceptibility alleles for T1D on the AH18.2 haplotype.
Assuntos
Mapeamento Cromossômico , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Haplótipos , Polimorfismo de Nucleotídeo Único , Idade de Início , Alelos , Estudos de Casos e Controles , Cromossomos Humanos , Diabetes Mellitus Tipo 1/imunologia , Feminino , Marcadores Genéticos , Genótipo , Antígenos HLA-B , Antígeno HLA-B18 , Homozigoto , Humanos , Masculino , Repetições de Microssatélites , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , EspanhaRESUMO
Interleukin-10 (IL10), an anti-inflammatory cytokine, has been implicated in a variety of immune- and inflammatory-related diseases. We investigated the following SNPs: -1082, -819, -592 in the promoter region of IL10 in a normal (control) population and selected diseases: breast cancer (BrCa), systemic lupus erythematosus (SLE), and B-cell chronic lymphocytic leukemia (B-CLL) by denaturing high-performance liquid chromatography (DHPLC) and found distinct genotype and haplotype patterns. DHPLC was performed using the Transgenomic WAVE instrument, a mutational discovery tool that allows for high throughout analysis of SNPs. The principle of DHPLC is based on separation of homo- and heteroduplex formation of individual polymerase chain reaction products at specific melting temperatures and set gradients. The melting temperature selected for each SNP was based on size and sequence of the polymerase chain reaction product (for -1082, 57 degrees C; for -819, 58 degrees C; and for -592, 59.2 degrees C). Before fragment mutational analysis, all samples were denatured at 95 degrees C and slowly reannealed to allow for reassociation of different strands. Heteroduplex samples were easily distinguished from homoduplex samples. In order to identify wild type from homozygous mutant, two homoduplex polymerase chain reaction samples had to be mixed together, denatured at 95 degrees C and reannealed. The homozygous mutant, when combined with wild type, displayed a double peak on chromatogram. Once distinct chromatograms were established for each of the SNPs and the nucleotide changes confirmed by sequencing, genotype and haplotype frequencies were tabulated for the groups studied.
Assuntos
Cromatografia Líquida de Alta Pressão , Interleucina-10/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Sequência de Bases , Neoplasias da Mama/genética , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Predisposição Genética para Doença , Humanos , Interleucina-10/biossíntese , Leucemia Linfocítica Crônica de Células B/genética , Lúpus Eritematoso Sistêmico/genética , Dados de Sequência MolecularRESUMO
The dietary phytochemical indole-3-carbinol (I3C) protects against cervical cancer in animal model studies and in human clinical trials. I3C and its physiologic condensation product diindolylmethane (DIM) also induce apoptosis of tumor cells in vitro and in vivo, suggesting that these phytochemicals might be useful as therapeutic agents as well as for cancer prevention. Deoxyribonucleic acid microarray studies on transformed keratinocytes and tumor cell lines exposed to pharmacologic concentrations of DIM in vitro are consistent with a cellular response to nutritional deprivation or disruptions in protein homeostasis such as endoplasmic reticulum (ER) stress. In this report we investigate whether specific stress response pathways are activated in tumor cells exposed to DIM and whether the ER stress response might contribute to DIM's cytotoxicity. Induction of the stress response genes GADD153, GADD34 and GADD45A, XBP-1, GRP78, GRP94, and asparagine synthase was documented by Western blot and real-time reverse transcriptase-polymerase chain reaction in C33A cervical cancer cells, and induction of a subset of these was also observed in cancer cell lines from breast (MCF-7) and prostate (DU145). The results are consistent with activation of more than 1 stress response pathway in C33A cells exposed to 75 microM DIM. Phosphorylation elF2alpha was rapidly and transiently increased, followed by elevated levels of ATF4 protein. Activation of IRE1alpha was indicated by a rapid increase in the stress-specific spliced form of XBP-1 messenger ribonucleic acid and a rapid and persistent phosphorylation of JNK1 and JNK2. Transcriptional activation dependent on an ATF6-XBP-1 binding site was detected by transient expression in MCF-7, C33A, and a transformed epithelial cell line (HaCaT); induction of the GADD153 (CHOP) promoter was also confirmed by transient expression. Cleavage of caspase 12 was observed in both DIM-treated and untreated C33A cells but did not correlate with cytotoxicity, whereas caspase 7 was cleaved at later times, coinciding with the onset of apoptosis. The results support the hypothesis that cytotoxic concentrations of DIM can activate cellular stress response pathways in vitro, including the ER stress response. Conversely, DIM was especially cytotoxic to stressed cells. Thapsigargin and tunicamycin, agents that induce ER stress, sensitized cells to the cytotoxic effects of DIM to differing degrees; nutrient limitation had a similar, but even more pronounced, effect. Because DIM toxicity in vitro is enhanced in cells undergoing nutritional deprivation and ER stress, it is possible that stressed cells in vivo, such as those within developing solid tumors, also have increased sensitivity to killing by DIM.
Assuntos
Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Fator 4 Ativador da Transcrição , Fator 6 Ativador da Transcrição , Antígenos de Diferenciação , Apoptose/fisiologia , Aspartato-Amônia Ligase/genética , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT/genética , Caspase 12 , Caspase 7 , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Proteínas de Ligação a DNA/genética , Desoxiglucose/farmacologia , Retículo Endoplasmático/fisiologia , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases , Expressão Gênica/genética , Proteínas de Choque Térmico HSP70/genética , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Leucina/deficiência , Luciferases/genética , Luciferases/metabolismo , Proteínas de Membrana/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Proteína Fosfatase 1 , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tapsigargina/farmacologia , Fator de Transcrição CHOP , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteína 1 de Ligação a X-Box , eIF-2 Quinase/metabolismoRESUMO
Diindolylmethane (DIM), a biologically active congener of indole-3-carbinol (I3C) derived from cruciferous vegetables, is a promising agent for the prevention of estrogen-sensitive cancers. Both DIM and estrogen affect transcription of genes by binding receptors, such as aryl hydrocarbon receptor (AhR) or estrogen receptors (ER). Gene regulation by DIM and estradiol (E2) can be very complex. While DIM typically binds the AhR, this complex can directly associate with the ER, recruit co-activators that bind to estrogen-responsive promoters, and activate transcription. Alternately, DIM can bind the ER directly. In this study, we have analyzed gene expression using microarray profiling and quantitative real time-polymerase chain reaction in MCF7 breast cancer cells treated with E2 (1 nM) or DIM (25 microM) alone or in combination for 16 h. The interplay of E2 and DIM was reflected in the expression of a subset of genes (<90) in which the combination of E2 and DIM acted either additively or antagonistically to alter gene expression.