Sequence of synthetic oligonucleotide primers used to characterize
1. Introduction
The genus
In 1984, Warren and Marshall isolated a bacterium from human stomachs in some patients with gastrointestinal disorders [2]. The organism was initially named “
The prevalence of
Even though its transmission pathways are not completely clarified [18], the infection appears to be usually acquired during childhood and is characterized as being chronic [19]. Routes of
After colonization, all patients with
Colonization with
However, the propensity to develop disease is an aspect that remains unclear, but may depend on host characteristics, particular bacterial factors (virulence of the infecting strains), or to the specific interactions between host and microbe, besides the environmental factors [24].
In fact, the interest in
The vast majority of gastric cancers are adenocarcinomas, which can be prevalently divided into two types, the intestinal and the diffuse [26], which corresponds, respectively, to the well-differentiated type and to the poorly-differentiated type, in the Japanese classification [22]. In contrast to the diffuse type often associated with familial distribution and developed in the stomach following chronic inflammation, especially in the cardia [27], intestinal type adenocarcinomas are generally thought to be preceded by a sequence of precursor lesions [28]. The basic components of this process are chronic inflammation of the gastric mucosa, which slowly progresses through the premalignant stages of atrophic gastritis, intestinal metaplasia and dysplasia to gastric cancer [29] that are most frequently localized in the antrum [30].
Patients diagnosed in an early stage of the cancer (Figure 3) present an excellent prognosis, with a five-year survival rate greater than 90%. In cases with advanced lesions, gastric cancer carries a poor prognosis, with an overall five-year survival rate of less than 20% [31].
With respect specifically to the bacterium strains,
The cagPAI is a 40 kb region of chromosomal DNA encoding approximately 31 genes that forms a type IV secretion system and can be divided into two regions, cag I and cag II, according to a novel insertion sequence [35]. This secretion system forms a pilus that delivers CagA, an oncoprotein, into the cytosol of gastric epithelial cells through a rigid needle structure covered by CagY, a VirB10-homologous protein and CagT, a Virb7-homologous protein, at the base [36-38].
Upon delivery into host cells by the
Consequently, the presence of the cagA gene has been associated with higher grades of inflammation, which may lead to the development of the most severe gastrointestinal diseases, such as peptic ulcer [47-49] and gastric cancer [50-54]. In Western countries, it has been reported that individuals infected with cagA-positive strains of
Furthermore, cagA is a polymorphic gene that presents different numbers of repeat sequences located in its 3’ region. Each repeat region of the CagA protein contains Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, including a tyrosine phosphorylation site. The first repeat region is commonly named as EPIYA-A and EPIYA-B segments and the second repeat region is named EPIYA-C or EPIYA-D segments, for Western and East Asian strains, respectively [57]. Despite the know variability in the N-terminal cagA gene and other cagPAI genes, there has been limited information concerning clinical relevance of genetic variants outside the EPIYAs [58].
Together with cagA gene, cagT gene is found at the base of the outgrowing pilus of the type IV secretion system and is supposed to be responsible for binding to a cellular receptor to induce interleukine-8 secretion and eject CagA [37]. The cagT gene has also been associated with higher degrees of inflammation, being encountered in
Thus, considering each gene, there are two types of clinical
As gastric cancer still ranks as a leading cause of cancer-related deaths in many parts of the world, the aims of the present study were to investigate the presence of cagA and cagT genes in Brazilian patients with early and advanced distal type intestinal adenocarcinoma, in order to determine the general incidence of this gene in gastric cancer and to compare the results obtained between the two stages of the disease, trying to understand if the cagA and the cagT genes and/or the genotype cagAcagT could be important to the development of one or other stage of distal intestinal type gastric adenocarcinoma in Brazil.
2. Clinical samples, methods and results
2.1. Clinical Samples
The present study was carried out utilizing clinical samples of
2.2. Methods
2.2.1. DNA extraction
Paraffin wax-embedded tissue DNA extraction was performed by carrying out the pre-extraction treatment of fixed tissues, using xylene and ethanol washes for paraffin removal. Subsequently, successive steps using proteinase K, phenol, chloroform and isoamyl alcohol were carried out, in order to isolate and purify the DNA [59]. Quantification of the extracted DNA and polymerase chain reaction (PCR) for human betaglobin gene [60] were carried out in order to guarantee the quality of this research.
2.2.2. PCR for urease C gene, cagA gene and cagT gene
Primers pairs for all the genes are described in Table 1, as well as the length of the fragments amplified for each reaction, urease C [61], cagA [62], and cagT [63]. PCR for urease C gene was performed with the aim to identify the bacterium DNA in samples.
After amplification, each PCR product was submitted to eletrophoresis on a 2.0% agarose gel stained by ethidium bromide with a 0.5X tris-acetate-EDTA buffer. A 100-bp ladder was used as standard.
Then, for each specific reaction, after being tested positive for urease C gene, products obtained were classified in cagA positive or negative and cagT positive or negative.
betaglobin |
+ - |
ACA CAA CTG TGT TCA CTA GC CAA CTT CAT CCA CGT TTC ACC |
110 |
urease C | + - |
AAG CTT TTA GGG GTG TTA GGG GTT T AAG CTT ACT TTC TAA CAC TAA CGC |
294 |
cagA | + - |
GAT AAC AGG CAA GCT TTT GAG G CTG CAA AAG ATT GTT TGG CAG A |
349 |
cagT | + - |
CCA TGT TTA TAC GCC TGT GT CAT CAC CAC ACC CTT TTG AT |
301 |
2.2.3. Statistical Analysis
After the amplification reactions, the results were analysed by a chi-square test at the Statistical Service of the Faculty of Medical Sciences, State University of Campinas (UNICAMP), with the
2.3. Results
PCR for the urease C gene of
As regards to cagT gene of
|
|
|
|
|
urease C |
31 (100.0%) | 58 (100.0%) | 89 (100.0%) | |
cagA positive cagA negative |
19 (61.3%) 12 (38.7%) |
48 (82.8%) 10 (17.2%) |
89 (100.0%) | 0.025* |
cagT positive cagT negative |
17 (54.8%) 14 (45.2%) |
38 (65.5%) 20 (34.5%) |
89 (100.0%) | 0.323 |
Total |
31 (100.0%) | 58 (100.0%) | 89 (100.0%) |
After the individual analysis, a table with absolute frequencies (n) and percentages (%) was made in order to compare the results individually for each gene and the genotypes combinations (Table 3; Table 4, respectively). There were 75.28% of positive cases for cagA gene and 61.80% of positive cases for cagT gene (table 3). As regards to cagAcagT, the most frequent genotype was cagA+cagT+, that was presented in 52.81% of the samples (table 4). The second most frequent genotype was cagA+cagT-, presented in 22.47% of the samples (table 4).
Subsequently, the combinations of genotypes were made and then compared between the two groups of patients, with early and advanced gastric adenocarcinoma. Although the most frequent genotype (cagA+cagT+) was the same in both groups of patiens, there was found a statistically significant difference between the groups, with cagA+cagT+ in advanced gastric cancer group (62.07%, 36 cases) and with cagA-cagT+ in early gastric cancer group (19.35%, 6 cases), p=0.027 (table 5).
|
|
|
cagA negative | 22 | 24.72 |
cagA positive | 67 | 75.28 |
cagT negative | 34 | 38.20 |
cagT positive | 55 | 61.80 |
|
|
|
cagA cagT | ||
negative negative | 14 | 15.73 |
negative positive | 8 | 8.99 |
positive negative | 20 | 22.47 |
positive positive | 47 | 52.81 |
|
|
||
negative negative | 8 13.79 |
6 19.35 |
14 |
negative positive | 2 3.45 |
6 |
8 |
positive negative | 12 20.69 |
8 25.81 |
20 |
positive positive | 36 |
11 35.48 |
47 |
|
58 | 31 | 89 |
3. Conclusion
Gastritis is the most common illness associated to the stomach and can be considered as the beginning of different complication that may led to peptic ulcers disease and gastric adenocarcinoma. Specifically concerning to gastric cancer, the understanding of the development of this disease has advanced considerably in recent decades, especially as regards to the role of
The risk of development of this disease is also related to genetic characteristics of the host and environmental factors, which, associated with specific bacterium strain characteristics, influence the severity of the chronic inflammatory response [64]. Thus, although infection with
Other question that remains unclear, and that can be associated with these bacterium virulence factors, is why some tumors remain in the early stage and others are in advanced stage almost always in a short period of time? Are the different strains of
In the present study we tried to understand some of these questions considering only one aspect of the development of the cancer, which were
As regards to cagA gene, in an isolated analysis, our results were similar to those of in which strains cagA positive were related to the development of gastric cancer. cagA positive strains tend to be more virulent and induce higher levels of expression of cytokines such as interleukin 1b and 8 [66]. Some studies have shown that patients with strains that express CagA are three times more likely to develop gastric cancer (52,57,67] than those infected with cagA negative strains [56,68]. Besides, other study demonstrated that strains that express CagA are three times more likely to develop advanced gastric cancer than the early stage [54]. In the present study the same characteristics were observed and patients infected with cagA positive strains demonstrated a high risk of advanced gastric cancer development (p=0.025; OR=3.023, 95%CI). Besides, studies conducted in Western countries [69,70] and in Asian countries [71] reported that the most patients with gastric cancer are infected with
One limitation for the comparisons of our results with others were that our study classified the two stages of the gastric cancer, the early and the advanced ones, and the most part of the studies consider only the gastric cancer disease, without classifying it in early or advanced stages. Besides, it was not our aim the study of the polymorphism of this gene, which are related to the EPIYA motifs, which can be important if we considerer the cagA gene in an isolated analysis, even though there has been limited information concerning clinical relevance of genetic variants outside the EPIYAs.
With our results, we can conclude that cagA gene is important for the development both of early and advanced gastric cancer, because it was presented in the most part of the strains in the two stages of the disease. Nevertheless, we can not conclude if it is important for the change of one stage in other (early in advanced stage) and we have also to consider that we do not really know if the bacterium presence is important or crucial after the beginning of the development of the disease. Some studies have demonstrated that the eradication of
Like the cagA gene, the cagT also belongs to the cagPAI and it is assumed that is related to the type IV secretion system, responsible for binding to cell receptors and inducing the release of interleukin-8 and also by ejecting the CagA protein [37]. The cagT gene has been linked to the development of peptic ulcer, and strains with the absence of this gene were generally related to chronic gastritis [63]. In this study, values were not found to be statistically significant between the two studied groups (p=0.323). However, the cagT gene was found, in the group as a whole, in 61.8% of the samples, which reflects an important result, showing that this gene, like cagA, or acting together with cagA, may be related to the gastric cancer development.
As regards to the comparison of the genotype cagAcagT, we reported again as a limitation for our study the fact that there are no studies that consider the early and the advanced stages of gastric cancer separately, so there were no relevant studies with which to compare the results from different stages of the same disease, gastric cancer. Further, we considered it unnecessary to make a comparison between these results and possible results from healthy individuals, because our aim was exactly to compare the strains presented in two stages of the same disease and not between the strains in the disease and in healthy volunteers.
For both groups, advanced and early, the most frequent genotype was cagA+cagT+ (62.07% and 35.48%, respectively). However, when comparison between the two groups was made, we found a statistically significance concerning the cagA+cagT+ and cagA-cagT+ strains (p=0.027). It can be considered very important because even though the genes cagA+ and cagT+ status were more frequent in both groups, the genotype cagA-cagT+ occurred with a higher frequency in the early gastric cancer group (advanced = 3.45%; early = 19.35%). Moreover, the genotype cagA+cagT+ occurred with a higher frequency in the advanced cancer group, a result that can be considered important too (advanced = 62.07%; early = 35.48%). We can suggest, with these results, that cagA gene positivity, independently of its polymorphisms, can be considered an essential virulence factor for the development of most severe gastric diseases, as gastric cancer.
Finally, as told before, the understanding of gastric carcinogenesis has advanced considerably over the past decades, especially with regards to insights into the role of
Obviously, more studies are necessary to elucidate the
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