CagA phosphorylation EPIYA-C motifs and the vacA i genotype in Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil.

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric diseases. Virulence factors such as VacA and CagA have been shown to increase the risk of these diseases. Studies have suggested a causal role of CagA EPIYA-C in gastric carcinogenesis and this factor has been shown to be geographically diverse. We investigated the number of CagA EPIYA motifs and the vacA i genotypes in H. pylori strains from asymptomatic children. We included samples from 40 infected children (18 females and 22 males), extracted DNA directly from the gastric mucus/juice (obtained using the string procedure) and analysed the DNA using polymerase chain reaction and DNA sequencing. The vacA i1 genotype was present in 30 (75%) samples, the i2 allele was present in nine (22.5%) samples and both alleles were present in one (2.5%) sample. The cagA-positive samples showed distinct patterns in the 3’ variable region of cagA and 18 of the 30 (60%) strains contained 1 EPIYA-C motif, whereas 12 (40%) strains contained two EPIYA-C motifs. We confirmed that the studied population was colonised early by the most virulent H. pylori strains, as demonstrated by the high frequency of the vacA i1 allele and the high number of EPIYA-C motifs. Therefore, asymptomatic children from an urban community in Fortaleza in northeastern Brazil are frequently colonised with the most virulent H. pylori strains.

CagA phosphorylation EPIYA-C motifs and the vacA i genotype in Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric diseases. Virulence factors such as VacA and CagA have been shown to increase the risk of these diseases. Studies have suggested a causal role of CagA EPIYA-C in gastric carcinogenesis and this factor has been shown to be geographically diverse. We investigated the number of CagA EPIYA motifs and the vacA i genotypes in H. pylori strains from asymptomatic children. We included samples from 40 infected children (18 females and 22 males), extracted DNA directly from the gastric mucus/juice (obtained using the string procedure) and analysed the DNA using polymerase chain reaction and DNA sequencing. The vacA i1 genotype was present in 30 (75%) samples, the i2 allele was present in nine (22.5%) samples and both alleles were present in one (2.5%) sample. The cagA-positive samples showed distinct patterns in the 3’ variable region of cagA and 18 of the 30 (60%) strains contained 1 EPIYA-C motif, whereas 12 (40%) strains contained two EPIYA-C motifs. We confirmed that the studied population was colonised early by the most virulent H. pylori strains, as demonstrated by the high frequency of the vacA i1 allele and the high number of EPIYA-C motifs. Therefore, asymptomatic children from an urban community in Fortaleza in northeastern Brazil are frequently colonised with the most virulent H. pylori strains. .

Single nucleotide polymorphisms of Helicobacter pylori dupA that lead to premature stop codons.

BACKGROUND: The detection of the putative disease-specific Helicobacter pylori marker duodenal ulcer promoting gene A (dupA) is currently based on PCR detection of jhp0917 and jhp0918 that form the gene. However, mutations that lead to premature stop codons that split off the dupA leading to truncated products cannot be evaluated by PCR. METHODS: We directly sequence the complete dupA of 75 dupA-positive strains of H. pylori isolated from patients with gastritis (n = 26), duodenal ulcer (n = 29), and gastric carcinoma (n = 20), to search for frame-shifting mutations that lead to stop codon. RESULTS: Thirty-four strains had single nucleotide mutations in dupA that lead to premature stop codon creating smaller products than the predicted 1839 bp product and, for this reason, were considered as dupA-negative. Intact dupA was more frequently observed in strains isolated from duodenal ulcer patients (65.5%) than in patients with gastritis only (46.2%) or with gastric carcinoma (50%). In logistic analysis, the presence of the intact dupA independently associated with duodenal ulcer (OR = 5.06; 95% CI = 1.22-20.96, p = .02). CONCLUSION: We propose the primer walking methodology as a simple technique to sequence the gene. When we considered as dupA-positive only those strains that carry dupA gene without premature stop codons, the gene was associated with duodenal ulcer and, therefore, can be used as a marker for this disease in our population.

Relationship of the methylenetetrahydrofolate reductase C677T polymorphism with microsatellite instability and promoter hypermethylation in sporadic colorectal cancer.

The methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with the expression of a thermolabile enzyme with decreased activity that influences the pool of methyl-donor molecules. Several studies have reported an association between C677T polymorphism and susceptibility to colorectal cancer (CRC). Considering that methylation abnormalities appear to be important for the pathogenesis of CRC, we examined the correlation between the genotype of the MTHFR C677T polymorphism, hypermethylation of the promoter region of five relevant genes (DAPK, MGMT, hMLH1, p16(INK4a), and p14(ARF)), and microsatellite instability, in 106 patients with primary CRCs in Brazil. We did not find significant differences in the genotypic frequencies of the MTHFR C677T polymorphism when one or more loci were hypermethylated. However, we did find a significant excess of 677TT individuals among patients with CRC who had microsatellite instability. This strong association was independent of the methylation status of hMLH1 and of the biogeographical genomic ancestry of the patients. Although the mechanism responsible for the link between the C677T polymorphism and microsatellite instability was not apparent, this finding may provide a clue towards a better understanding of the pathogenesis of microsatellite instability in human colorectal cancer.

Colorectal cancer "methylator phenotype": fact or artifact?

It has been proposed that human colorectal tumors can be classified into two groups: one in which methylation is rare, and another with methylation of several loci associated with a "CpG island methylated phenotype (CIMP)," characterized by preferential proximal location in the colon, but otherwise poorly defined. There is considerable overlap between this putative methylator phenotype and the well-known mutator phenotype associated with microsatellite instability (MSI). We have examined hypermethylation of the promoter region of five genes (DAPK, MGMT, hMLH1, p16INK4a, and p14ARF) in 106 primary colorectal cancers. A graph depicting the frequency of methylated loci in the series of tumors showed a continuous, monotonically decreasing distribution quite different from the previously claimed discontinuity. We observed a significant association between the presence of three or more methylated loci and the proximal location of the tumors. However, if we remove from analysis the tumors with hMLH1 methylation or those with MSI, the significance vanishes, suggesting that the association between multiple methylations and proximal location was indirect due to the correlation with MSI. Thus, our data do not support the independent existence of the so-called methylator phenotype and suggest that it rather may represent a statistical artifact caused by confounding of associations.

Development and application of a multiplex PCR procedure for the detection of DNA methylation in colorectal cancer.

Cancers often exhibit aberrant methylation of gene promoter regions associated with loss of tumor suppressor and/or DNA repair gene function. Such methylation constitutes an excellent marker for the molecular detection of micro-metastases and the diagnosis of tumor recurrences. We have developed a multiplex methylation-specific PCR (MSP) procedure for rapid and simultaneous assessment of the methylation of 5 loci: the tumor suppressor genes p16INK4a, death-associated protein kinase (DAPK) and p14ARF, and the DNA repair genes hMLH1 and O6-methylguanine-DNA-methyltransferase (MGMT). This multiplex test uses one single PCR reaction and only one electrophoretic run. In 98 samples of colorectal cancer studied, methylation of MGMT, DAPK, p16, hMLH1 and p14 was present in 31, 20, 17, 16 and 14% of tumors, respectively. In 58% of the tumors at least one methylated gene was found. This multiplex MSP constitutes a simple and inexpensive method for screening of molecular signatures in colorectal cancer and can be used profitably before employing more expensive and complex techniques such as microarray testing.

Mining microorganism EST databases in the quest for new proteins.

Microorganisms with large genomes are commonly the subjects of single-round partial sequencing of cDNA, generating expressed sequence tags (ESTs). Usually there is a great distance between gene discovery by EST projects and submission of amino acid sequences to public databases. We analyzed the relationship between available ESTs and protein sequences and used the sequences available in the secondary database, clusters of orthologous groups (COG), to investigate ESTs from eight microorganisms of medical and/or economic relevance, selecting for candidate ESTs that may be further pursued for protein characterization. The organisms chosen were Paracoccidioides brasiliensis, Dictyostelium discoideum, Fusarium graminearum, Plasmodium yoelii, Magnaporthe grisea, Emericella nidulans, Chlamydomonas reinhardtii and Eimeria tenella, which have more than 10,000 ESTs available in dbEST. A total of 77,114 protein sequences from COG were used, corresponding to 3,201 distinct genes. At least 212 of these were capable of identifying candidate ESTs for further studies (E. tenella). This number was extended to over 700 candidate ESTs (C. reinhardtii, F. graminearum). Remarkably, even the organism that presents the highest number of ESTs corresponding to known proteins, P. yoelii, showed a considerable number of candidate ESTs for protein characterization (477). For some organisms, such as P. brasiliensis, M. grisea and F. graminearum, bioinformatics has allowed for automatic annotation of up to about 20% of the ESTs that did not correspond to proteins already characterized in the organism. In conclusion, 4093 ESTs from these eight organisms that are homologous to COG genes were selected as candidates for protein characterization.

Structural and functional analysis of an amplification containing a PGPA gene in a glucantime-resistant Leishmania (Viannia) guyanensis cell line.

Drug resistance is a complex phenomenon in Leishmania and commonly involves gene amplification. Active efflux and metal sequestration through a P-glycoprotein have been pointed to as the major mechanisms used by drug-resistant Leishmania. A gene amplification from a glucantime-resistant Leishmania (Viannia) guyanensis cell line was characterised in an attempt to understand the mechanism of metal resistance in this pathogenic species. We show that the amplification is present as an extrachromosomal amplicon of 30 kb and contains a PGPA gene ( LgPGPA), which is overexpressed in the resistant line as shown by Northern and Western blot analyses. In addition, we gathered evidence from transfection experiments for the role of the LgPGPA gene in oxyanion resistance in L. (V.) guyanensis. Our work indicates that, in this pathogenic New World Leishmania species, amplification of the PGPA gene is the major determinant in oxyanion resistance.