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- The D1822V APC polymorphism interacts with fat, calcium, and fiber intakes in modulating the risk of colorectal cancer in Portuguese personsPublication . Guerreiro, Catarina Sousa; Cravo, Marília; Brito, Miguel; Vidal, Pedro L.; Fidalgo, Paulo O.; Leitão, Carlos NobreBackground - Both genetic and environmental factors affect the risk of colorectal cancer (CRC). Objective - We aimed to examine the interaction between the D1822V polymorphism of the APC gene and dietary intake in persons with CRC. Design - Persons with CRC (n = 196) and 200 healthy volunteers, matched for age and sex in a case-control study, were evaluated with respect to nutritional status and lifestyle factors and for the D1822V polymorphism. Results - No significant differences were observed in energy and macronutrient intakes. Cases had significantly (P < 0.05) lower intakes of carotenes, vitamins C and E, folate, and calcium than did controls. Fiber intake was significantly (P = 0.004) lower in cases than in controls, whereas alcohol consumption was associated with a 2-fold risk of CRC. In addition, cases were significantly (P = 0.001) more likely than were controls to be sedentary. The homozygous variant for the APC gene (VV) was found in 4.6% of cases and in 3.5% of controls. Examination of the potential interactions between diet and genotype found that a high cholesterol intake was associated with a greater risk of colorectal cancer only in noncarriers (DD) of the D1822V APC allele (odds ratio: 1.66; 95% CI: 1.00, 2.76). In contrast, high fiber and calcium intakes were more markedly associated with a lower risk of CRC in patients carrying the polymorphic allele (DV/VV) (odds ratio: 0.50; 95% CI: 0.27, 0.94 for fiber; odds ratio: 0.51; 95% CI: 0.28, 0.93 for calcium) than in those without that allele. Conclusion - These results suggest a significant interaction between the D1822V polymorphism and the dietary intakes of cholesterol, calcium, and fiber for CRC risk.
- A STR mutation in a heteropaternal twin casePublication . Geada, Helena; Ribeiro, Teresa; Brito, Miguel; Espinheira, Rosa; Rolf, Burkhard; Hohoff, Carsten; Brinkmann, BerndA heteropaternal male twin case with two men being alleged fathers was investigated as requested by the Court. Up to 37 PCR-based polymorphic DNA systems were studied in this case which was complicated by a paternal ACTBP2 mutation detected in one twin. This is the first report on a STR mutation in a double paternity case where both biological fathers were indisputably identified. The STR systems enable the resolution of these complex genetic relationships even in a case where a mutation in one STR locus was encountered.
- Detecção e quantificação de soja geneticamente modificada em géneros alimentícios, comercializados em Portugal, para consumo humanoPublication . Faustino, Rita; Sousa, Ana; Loureiro, Marta; Mendes, Lino; Brito, MiguelA biotecnologia permite modificar geneticamente plantas e animais, dando origem a organismos geneticamente modificados (OGM). Os benefícios e potenciais riscos relacionados com os transgénicos são controversos, pelo que o consumidor tem direito a decidir sobre o seu consumo. A soja é a maior representante do mercado global de transgénicos, tornando-se importante a sua monitorização. O objectivo do presente trabalho é detectar e quantificar soja geneticamente modificada em géneros alimentícios. Para tal, 11 amostras de dois lotes diferentes, de uma mesma marca, de géneros alimentícios contendo soja, para consumo humano, foram analisadas pelo método de PCR em tempo real para detecção e quantificação de DNA transgénico. Do total das amostras analisadas, 7 resultaram negativas (63,6%) e 4 positivas (36,4%) para a detecção de DNA de soja transgénica, correspondentes a 3 alimentos: leite, farinha e granulado de soja. As quantidades encontradas variaram entre 0,07% (granulado de soja) e 0,22% (farinha de soja), sugestivas de contaminação ambiental por serem inferiores a 0,4%. Este trabalho evidencia a necessidade do controlo dos alimentos, mostrando ainda a possibilidade de aplicação da PCR em tempo real na detecção e quantificação de OGM mesmo em alimentos processados.
- Translation termination and protein folding pathway genes are not correlated in gastric cancerPublication . Malta-Vacas, Joana; Nolasco, Sofia; Monteiro, Carolino; Soares, Helena; Brito, MiguelBackground: The eukaryotic release factor 3 (eRF3) has been shown to affect both tubulin and actin cytoskeleton, suggesting a role in cytoskeleton assembly, mitotic spindle formation and chromosome segregation. Also, direct interactions between eRF3 and subunits of the cytosolic chaperonin CCT have been described. Moreover, both eRF3a and CCT subunits have been described to be up-regulated in cancer tissues. Our aim was to evaluate the hypothesis that eRF3 expression levels are correlated with the expression of genes encoding proteins involved in the tubulin folding pathways. Methods: Relative expression levels of eRF1, eRF3a/GSPT1, PFDN4, CCT2, CCT4, and TBCA genes in tumour samples relative to their adjacent normal tissues were investigated using real time-polymerase chain reaction in 20 gastric cancer patients. Results: The expression levels of eRF3a/GSPT1 were not correlated with the expression levels of the other genes studied. However, significant correlations were detected between the other genes, both within intestinal and diffuse type tumours. Conclusions: eRF3a/GSPT1 expression at the mRNA level is independent from both cell translation rates and from the expression of the genes involved in tubulin-folding pathways. The differences in the patterns of expression of the genes studied support the hypothesis of genetically independent pathways in the origin of intestinal and diffuse type gastric tumours.
- Fatty acids, IL6, and TNFα polymorphisms: an example of nutrigenetics in Crohn's diseasePublication . Guerreiro, Catarina Sousa; Ferreira, Paula; Tavares, Lourdes; Santos, Paula Moura dos; Neves, Manuela; Brito, Miguel; Cravo, MaríliaObjectives - The aim of this work was to study the interaction between genetic polymorphisms (single-nucleotide polymorphisms, SNPs) of pro- and anti-inflammatory cytokines and fat intake on the risk of developing Crohn's disease (CD) or modifying disease activity. Methods - Seven SNPs in interleukin 1 (IL1), tumor necrosis factor alpha (TNFalpha), lymphotoxin alpha (LTalpha), and IL6 genes were analyzed in 116 controls and 99 patients with CD. The type of fat intake was evaluated, and the interaction between SNPs and dietary fat in modulating disease activity was analyzed. Results - Individuals who were homozygous for the IL6-174G/C polymorphism had a six-fold higher risk for CD (odds ratio (OR)=6.1; 95% confidence interval (95% CI)=1.9-19.4), whereas the TT genotype on the TNFalpha-857C/T polymorphism was associated with more active disease (OR=10.4; 95% CI=1.1-94.1). A high intake of total, saturated, and monounsaturated fats, as well as a higher ratio of n-6/n-3 polyunsaturated fatty acid (PUFA), was associated with a more active phenotype (P<0.05). Furthermore, there was an interaction between dietary fat intake and SNPs, with a high intake of saturated and monounsaturated fats being associated with active disease, mainly in patients carrying the variant alleles of the 857 TNFalpha polymorphism (OR=6.0, 95% CI=1.4-26.2; OR=5.17; 95% CI=1.4-19.2, respectively) and the 174 IL6 polymorphism (OR=2.95; 95% CI=1.0-9.1; OR=3.21; 95% CI=1.0-10.4, respectively). Finally, low intake of n-3 PUFA and high n-6/n-3 PUFA ratio in patients with the TNFalpha 857 polymorphism were associated with higher disease activity (OR=3.6; 95% CI=1.0-13.0; OR=5.92; 95% CI=1.3-26.5, respectively). Conclusions - These results show that different types of fat may interact with cytokine genotype, modulating disease activity.
- Differential expression of GSPT1 GGCn alleles in cancerPublication . Malta-Vacas, Joana; Ferreira, Paula; Monteiro, Carolino; Brito, MiguelThe human eukaryotic release factor 3a (eRF3a), encoded by the G1 to S phase transition 1 gene (GSPT1; alias eRF3a), is upregulated in various human cancers. GSPT1 contains a GGCn polymorphism in exon 1, encoding a polyglycine expansion in the N-terminal of the protein. The longer allele, GGC12, was previously shown to be associated to cancer. The GGC12 allele was present in 2.2% of colorectal cancer patients but was absent in Crohn disease patients and in the control group. Real-time quantitative RT-PCR analysis showed that the GGC12 allele was present at up to 10-fold higher transcription levels than the GGC10 allele (P < 0.001). No GSPT1 amplifications were detected, and there was no correlation between the length of the alleles and methylation levels of the CpG sites inside the GGC expansion. Using flow cytometry, we compared the levels of apoptosis and proliferation rates between cell lines with different genotypes, but detected no significant differences. Finally, we used a cytokinesis-block micronucleus assay to evaluate the frequency of micronuclei in the same cell lines. Cell lines with the longer alleles had higher frequencies of micronuclei in binucleated cells, which is probably a result of defects in mitotic spindle formation. Altogether, these findings indicate that GSPT1 should be considered a potential proto-oncogene.
- Polyglycine expansions in eRF3/GSPT1 are associated with gastric cancer susceptibilityPublication . Brito, Miguel; Malta-Vacas, Joana; Carmona, Bruno; Aires, C.; Costa, P.; Martins, A. P.; Ramos, S.; Conde, A. R.; Monteiro, C.Gastric cancer remains a major cause of death in the developed countries, and a large percentage is still genetically unexplained. Because of their major role in cell survival, mutations in translation factors and altered expression of these genes have been associated with cancer development. Apart from its role in translation termination, the eukaryotic translation release factor 3 (eRF3) is involved in several critical cellular processes, such as cell cycle regulation, cytoskeleton organization, and apoptosis. The aim of this study was to evaluate eRF3/GSPT1 gene as a potential genetic susceptibility associated locus for gastric cancer, analyzing a stable GGC expansion in exon 1 encoding a polyglycine tract in the N-terminal domain of the protein. DNA was obtained from 139 patients with gastric cancer and from 100 individuals of a healthy control population. The GGC expansion was amplified by PCR and the number of repeats determined by genotyping in an automatic sequencer. There are five known alleles encoding from 8 to 12 glycines. The most common allele encodes 10 glycines. The 12-Gly allele was detected exclusively in the cancer patients (allelic frequency = 5%). Regardless of the genotype, patients with the 12-Gly allele had a 20-fold increased risk for gastric cancer. We also detected a single-base alteration in the gene (G274T) although no correlation with cancer development has been found. Thus, our results show that the GGC expansion may have a potential role in regulating eRF3/GSPT1 expression and/or changing the protein function that can lead to gastric cancer development.
- Differential expression of the eukaryotic release factor 3 (eRF3/GSPT1) according to gastric cancer histological typesPublication . Malta-Vacas, Joana; Aires, C.; Costa, P.; Conde, A. R.; Ramos, S.; Martins, A. P.; Monteiro, C.; Brito, MiguelBackground: There are now several lines of evidence to suggest that protein synthesis and translation factors are involved in the regulation of cell proliferation and cancer development. Aims: To investigate gene expression patterns of eukaryotic releasing factor 3 (eRF3) in gastric cancer. Methods: RNA was prepared from 25 gastric tumour biopsies and adjacent non-neoplastic mucosa. Real time TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed to measure the relative gene expression levels. DNA was isolated from tumour and normal tissues and gene dosage was determined by a quantitative real time PCR using SYBR Green dye. Results: Different histological types of gastric tumours were analysed and nine of the 25 tumours revealed eRF3/GSPT1 overexpression; moreover, eight of the 12 intestinal type carcinomas analysed overexpressed the gene, whereas eRF3/GSPT1 was overexpressed in only one of the 10 diffuse type carcinomas (Kruskal-Wallis Test; p , 0.05). No correlation was found between ploidy and transcript expression levels of eRF3/GSPT1. Overexpression of eRF3/GSPT1 was not associated with increased translation rates because the upregulation of eRF3/GSPT1 did not correlate with increased eRF1 levels. Conclusions: Overexpression of eRF3/GSPT1 in intestinal type gastric tumours may lead to an increase in the translation efficiency of specific oncogenic transcripts. Alternatively, eRF3/GSPT1 may be involved in tumorigenesis as a result of its non-translational roles, namely (dis)regulating the cell cycle, apoptosis, or transcription.
- eRF3a/GSPT1 12-GGC allele increases the susceptibility for breast cancer developmentPublication . Malta-Vacas, Joana; Chauvin, Celine; Gonçalves, Lucília; Nazaré, Antónia; Carvalho, Carlos; Monteiro, Carolino; Bagrel, Denyse; Jean-Jean, Olivier; Brito, MiguelIt is now widely recognized that translation factors are involved in cancer development and that components of the translation machinery that are deregulated in cancer cells may become targets for cancer therapy. The eukaryotic Release Factor 3 (eRF3) is a GTPase that associates with eRF1 in a complex that mediates translation termination. eRF3a/GSPT1 first exon contains a (GGC)n expansion coding for proteins with different N-terminal extremities. Herein we show that the longer allele (12-GGC) is present in 5.1% (7/137) of the breast cancer patients analysed and is absent in the control population (0/135), corresponding to an increased risk for cancer development, as revealed by Odds Ratio analysis. mRNA quantification suggests that patients with the 12-GGC allele overexpress eRF3a/GSPT1 in tumor tissues relative to the normal adjacent tissues. However, using an in vivo assay for translation termination in HEK293 cells, we do not detect any difference in the activity of the eRF3a proteins encoded by the various eRF3a/GSPT1 alleles. Although the connection between the presence of eRF3a/GSPT1 12-GGC allele and tumorigenesis is still unknown, our data suggest that the presence of the 12-GGC allele provides a potential novel risk marker for various types of cancer.
- 5' and 3' UTR thymidylate synthase polymorphisms modulate the risk of colorectal cancer independently of the intake of methyl group donorsPublication . Carmona, Bruno; Guerreiro, Catarina Sousa; Cravo, Marília; Leitão, Carlos Nobre; Brito, MiguelThymidylate synthase, as a rate-limiting step in DNA synthesis, catalyses the conversion of dUMP into dTMP using 5,10-methylenotetrahydrofolate as the methyl donor. Two polymorphisms have been described in this gene: a repeat polymorphism in the 5' promoter enhancer region (3R versus 2R) and a 6 bp deletion in the 3' unstranslated region. Both of these may affect protein levels. The present case control study was aimed at investigating the influence of these two polymorphisms on the development of colorectal cancer (CRC), as well as their potential interaction with folate, vitamin B6 and vitamin B12 intake. A total of 196 cases and 200 controls, matched for age and sex distribution, were included in the study. No association was found between CRC and the 28 bp repeat polymorphism, but it was observed that individuals with the 6 bp/del and del/del genotypes had a significantly lower risk of developing the disease (OR=0.47; 95% CI 0.30-0.72). A combined genotype (2R/2R; 6 bp/del+del/del) was also found, which was associated with an even lower risk of developing of the disease (OR=0.42; 95% CI 0.26-0.69). No significant interaction between these polymorphisms and vitamin intake was observed. These results indicate for the first time that the 6 bp/del allele might be a protective factor in the development of CRC, independent of the intake of methyl group donors.