J Med Microbiol 57 (2008), 554-562; DOI: 10.1099/jmm.0.47776-0
© 2008 Society for General Microbiology
ISSN 1473-5644
dupA as a risk determinant in Helicobacter pylori infection
Masoumeh Douraghi1,2,
Marjan Mohammadi1,
Akbar Oghalaie1,
Afshin Abdirad3,
Mohammad Ali Mohagheghi3,
Mahmoud Eshagh Hosseini4,
Hojat Zeraati5,
Amir Ghasemi2,
Maryam Esmaieli1 and
Nazanin Mohajerani1
1 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran, 13164
2 Department of Pathobiology, School of Public Health, Medical Sciences/University of Tehran, Tehran, Iran
3 Cancer Research Center, Medical Sciences/University of Tehran, Tehran, Iran
4 Endoscopy Unit, Amiralam Hospital, Medical Sciences/University of Tehran, Tehran, Iran
5 Department of Biostatistics, School of Public Health, Medical Sciences/University of Tehran, Tehran, Iran
Correspondence
Marjan Mohammadi
marjan{at}pasteur.ac.ir
or
mmohammadi{at}yahoo.com
Received 20 November 2007
Accepted 6 January 2008
The Helicobacter pylori duodenal ulcer promoting (dupA) gene has been previously described as a risk marker for duodenal ulcer (DU) development and a protective factor against gastric cancer (GC). Recent studies which have assessed the application of dupA in the prediction of clinical outcomes have been controversial. In the current study, the association of dupA with the clinical outcomes and histopathological changes following H. pylori infection was evaluated in Iranian patients. A total of 157 H. pylori-infected patients with DU (n=30), gastric ulcer (n=23), gastritis (n=68) or GC (n=36) were assessed. The presence of jhp0917 and jhp0918 genes was determined by gene specific PCR. Gastric histopathological changes were recorded according to the updated Sydney system. Seventy-eight (49.7 %) and 71 (45.2 %) of the 157 tested strains, respectively, were positive and negative for both genes. The remaining 8 (5.09 %) of the 157 strains were jhp0917-positive/jhp0918-negative. Univariate analysis showed inverse associations between dupA and histological features including dysplasia as the penultimate stage of GC and lymphoid follicles as a consequence of relatively long-standing H. pylori-associated gastritis. The degrees of nucleotide sequence identity of Iranian strains to Colombian, Brazilian and Indian strains ranged from 86.1 to 100 % for the aligned regions of jhp0917, from 88 to 98.8 % for jhp0918 and from 93.4 to 99.5 % for the partial sequences of the dupA gene. Despite the fact that possession of the dupA gene showed no association with any disease category in our population as reported in several other countries, association of dupA-negative strains of H. pylori with pre-malignant lesions calls for additional studies to evaluate the role of this gene as a protective marker against GC.
Abbreviations: DU, duodenal ulcer; GC, gastric cancer; GU, gastric ulcer.
The GenBank/EMBL/DDBJ accession numbers for the target genes in this study are DQ865230, DQ865231, DQ865232, DQ865233, DQ865234, DQ865235, DQ865236, DQ865237, EF076755 and EF076756.
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INTRODUCTION
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Helicobacter pylori colonizes the gastric mucosa of humans, where it can establish a long-term infection resulting in active chronic gastritis, which may result in mutually exclusive outcomes of peptic ulcer disease versus gastric cancer (GC) (Blaser, 1997; Crabtree et al., 1991; Weel et al., 1996; Parsonnet et al., 1997; Figueiredo et al., 2001; Graham, 1989; Sipponen & Stolte, 1997; McColl et al., 2000; Uemura et al., 2001). Several virulence-associated genes of H. pylori, such as cagA, vacA, babA2 and oipA, are believed to play a major role in the associated clinical picture. Numerous studies have provided new insights into the role of these putative virulence factors in gastroduodenal pathogenesis (Covacci et al., 1993; Atherton et al., 1995; Censini et al., 1996; Gerhard et al., 1999; Yamaoka et al., 1999, 2000, 2002; Miehlke et al., 2000; Prinz et al., 2001; Yu et al., 2002; Azuma et al., 2002; Zambon et al., 2003; Argent et al., 2004). However, none of the mentioned virulence markers have demonstrated discriminating roles in the development of peptic ulcer disease versus GC. Genomic-sequence comparison of two unrelated isolates of H. pylori (26695 and J99) indicates the presence of several regions of exceptionally distinct G+C content. A large region of approximately 45 kb in J99 and 68 kb in 26695 with a G+C content lower than that of the rest of the genome has been termed the ‘plasticity zone’. This region includes half of the strain-specific open reading frames (ORFs) and is composed of 48 % and 46 % unique genes in strains J99 and 26695, respectively. Among the 38 ORFs in the J99 plasticity zone (jhp914–jhp951), 33 are missing in strain 26695 (Tomb et al., 1997; Alm et al., 1999; Alm & Trust, 1999). The jhp0917 and jhp0918 genes, which form one continuous gene, were studied by Lu et al. (2005) and described as a risk marker for development of duodenal ulcer (DU) and a protective factor against GC in strains isolated from Japan, Korea and Colombia. This continuous gene was thus referred to as the duodenal ulcer promoting (dupA) gene, and was found to be more prevalent in DU patients and associated with a reduced risk for development of gastric atrophy and cancer in these populations. Conversely, dupA genotyping by Argent et al. (2007) of four other populations, from Belgium, South Africa, China and the United States, showed no association of this gene with DU, but suggested an association with GC. Gomes et al. (2007) also negated any association between dupA and either of the two disparate clinical outcomes in Brazilian patients. In contrast, a recent report from India demonstrated a significant discriminative role for dupA as a virulence marker of DUs (Arachchi et al., 2007).
This study was thus undertaken to clarify the status of H. pylori dupA as a promoting marker for DU or a protective marker against GC, as well as the association of dupA with histopathological lesions in Iranian dyspeptic patients.
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METHODS
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Subjects and bacterial isolates.
The study population consisted of 157 H. pylori-infected patients with upper gastrointestinal disorders who were referred for endoscopy or gastric resection and were composed of the following groups: (i) 30 DU patients; (ii) 23 gastric ulcer (GU) patients; (iii) 68 gastritis patients; and (iv) 36 GC patients (21 cardia and 13 non-cardia type plus 2 with total involvement of the stomach). Gastritis was defined as an inflammatory condition of the gastric mucosa with no peptic ulcer or GC. Informed consent was obtained from every patient prior to sample collection, which was performed according to standard protocols approved by the local ethical committee.
H. pylori strains were primarily isolated from two antral biopsy specimens. Tissue samples were homogenized and plated onto H. pylori Specific Peptone Agar medium. Incubation was performed at 37 °C in a microaerophilic atmosphere (10 % CO2, 5 % O2 and 85 % N2) for up to 7 days. Identification was based on morphology under Gram staining and biochemical tests including urease and catalase tests.
DNA extraction and PCR.
Briefly, bacterial genomic DNA was extracted by incubating bacterial pellets in 50 mM NaOH at 100 °C for 20 min, followed by 10 min incubation in 1 M Tris/HCl (pH 7.5). The supernatants containing genomic DNA were used as the template for PCR amplification. The ureC gene was primarily amplified to confirm the identity of the isolated H. pylori strains (Labigne et al., 1991). This was followed by amplification of the jhp0917 and jhp0918 genes using specific primers (Lu et al., 2005). Tested strains were considered dupA-positive or -negative if both genes were present or absent simultaneously. Every PCR was repeated to confirm the results. In order to confirm the base insertion downstream of nucleotide 1385, partial dupA genes of 10 randomly selected strains (jhp0917/jhp0918-positive) were amplified using JHP0917F and JHP0918R as the forward and reverse primers, respectively (described by Lu et al., 2005), yielding a 1260 bp fragment. Amplification of the target genes was carried out in a total volume of 20 µl containing 2 µl 10x PCR buffer (Fermentas), 1.5 mM MgCl2, 0.5 µl of each primer (25 pmol µl–1), 0.2 mM deoxynucleotide and 0.5 U Taq polymerase. The cycling programmes were preceded by 4 min at 94 °C and consisted of 35 cycles of 94 °C for 50 s, 60 °C for 50 s and 72 °C for 50 s, followed by a final extension step at 72 °C for 4 min.
Sequence analysis.
The amplified products of jhp0917, jhp0918 and dupA were cut out of the agarose gel and purified using the standard phenol/chloroform procedure. The purified PCR products were sequenced using an automated sequencer. The obtained partial jhp0917, jhp0918 and dupA gene sequences were deposited in GenBank and aligned with similar gene segments of other reported gene sequences (Table 1
). Sequence analysis was performed with the BioEdit (version 5.0.6) and CLUSTAL_X (1.8) sequence alignment programs. The entire sequences of different strains were primarily aligned, followed by analysis of every sequence in comparison to that of the C142 strain (Table 1). Finally, the regions which were not common between the sequences of other strains were omitted and the sequence identity matrix for shorter fragments of various strains which matched those of strain C142 was generated.
Histopathological studies.
Multiple gastric biopsies were fixed in 10 % formalin and embedded in paraffin wax by routine methods. An expert pathologist blinded to the clinical diagnosis and characteristics of the infecting H. pylori strains examined the sections under haematoxylin and eosin and Giemsa staining for histopathological changes and the intensity of the colonizing H. pylori, respectively. The presence or absence of each histological feature, including chronic gastritis, neutrophil infiltration, lymphoid follicles, atrophy, intestinal metaplasia, dysplasia and colonization of H. pylori, were recorded.
Statistical analysis.
Data were analysed using the SPSS (version 11.5) program. For univariate analysis, the 
2 test and Fisher's exact test were used. The presence or absence of each pathological index was evaluated with respect to the possession of dupA by the infecting H. pylori strain, using the 2 test. A kappa test was used to determine the association and accordance between the presence of dupA and other virulence markers, including cagA and vacA. A univariate logistic regression analysis was performed to determine the risk of possession of dupA in relation to the clinical outcome. A P value of 
0.05 was considered statistically significant.
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RESULTS AND DISCUSSION
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Distribution of dupA
Seventy-eight of the 157 tested strains (49.7 %) were positive for both the jhp0917 and jhp0918 genes and thus considered dupA-positive. The prevalence of these genes in H. pylori strains in Iran compared to that in other studied populations is presented in Fig. 1.
Linkage of jhp0917 and jhp0918
The jhp0917 and jhp0918 genes were found in 86 (54.8 %) and 78 (49.7 %) of the H. pylori strains, respectively. Seventy-eight (49.7 %) strains were positive for both genes, whereas 71 (45.2 %) were negative. The presence of the jhp0917 and jhp0918 genes was strongly linked (P <0.001) with a small number of strains (5.09 %) possessing the jhp0917-positive/jhp0918-negative genotype. A jhp0917-negative/jhp0918-positive genotype was not detected.
Association between dupA and clinical outcomes
The distribution of H. pylori dupA-positive and -negative strains in patients with different clinical diagnoses is listed in Table 2. The prevalence of dupA in DU patients (50 %) was slightly higher than that in non-cardia GC patients (46.2 %) but did not reach statistical significance. The distribution of dupA in relation to clinical outcomes in Iran compared to that in other countries is depicted in Table 3. A uniform distribution (approx. 50 %) of dupA was found in all Iranian patient categories as was seen in Brazil and South Africa. The prevalence of dupA in DU patients and non-cardia GC patients from different countries ranged from 18 to 92 % and from 6 to 100 %, respectively. Univariate logistic regression analysis indicated that possession of dupA was not a promoting or protective determinant for any of the disease categories in our subjects.
dupA gene status and gastric histology
We also evaluated whether the presence of dupA was associated with histopathology indices. Univariate analysis demonstrated that possession of dupA was inversely associated with dysplasia such that 83.3 % of dysplasia-positive patients were colonized with dupA-negative strains (P <0.05) whereas only 30.2 % of dysplasia-negative patients harboured these strains (Table 4). The presence of lymphoid follicles was also inversely associated with dupA-positive strains (P <0.05), taking into account that 81.3 % of patients with no lymphoid follicles possessed dupA-positive strains whereas only 52.1 % of patients with lymphoid follicles were infected with these strains. When histological indices were separately studied in each disease category, the same associations existed but with peri-tumoral dysplasia in GC patients (P <0.05) and lymphoid follicles in gastritis patients (P <0.05) (Table 4). In this analysis, 90 % of patients who lacked neutrophil infiltration carried dupA-positive strains in comparison to 56 % of patients with neutrophil infiltration. Nevertheless, this phenomenon did not reach statistical significance. The presence of the dupA gene was not, however, related to chronic gastritis, atrophy, intestinal metaplasia or the presence of H. pylori (Table 4).
Association of dupA and major virulence markers of H. pylori
Forty-nine per cent of cagA-positive strains and 57.9 % of cagA-negative strains were dupA-positive. The presence of dupA was thus not associated with the presence of cagA in any patient category except for patients suffering from DUs (P <0.05). Forty-eight per cent of vacA s1 strains, 60 % of vacA s2 strains, 56.6 % of vacA m1 strains and 47.4 % of vacA m2 strains were dupA-positive. Collectively, neither kappa agreement nor chi-square analysis showed any statistically significant association between dupA status and cagA status or vacA polymorphisms. The same held true when the status of cagA and vacA was independently evaluated.
Comparative analysis of jhp0917, jhp0918 and dupA sequences
The nucleotide sequences of jhp0917, jhp0918 and dupA of six Iranian strains were aligned with the sequences of the corresponding genes in J99, C142 and ten Brazilian and three Indian strains. In comparison with C142 and the Brazilian and Indian strains, the nucleotide sequence similarity of Iranian strains ranged from 86.1 to 100 % for jhp0917, from 88 to 98.8 % for jhp0918 and from 93.4 to 99.5 % for dupA. The sequence similarities are presented in Tables 5, 6 and 7. This analysis revealed a significant similarity (>93 %) in partial sequences of dupA among different studied strains (Table 7). There was a ‘C’ insertion in a GC strain (Iran-5) and a ‘T’ insertion in a DU strain (Iran-6) downstream of nucleotide 1385. This is in accordance with every other studied strain except for J99, Brazil-4, Brazil-7 and Brazil-9 (Fig. 2).
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Table 5. Nucleotide similarity (%) of partial sequences of the jhp0917 gene
The accession numbers and the aligned positions are as indicated in Table 1 .
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Table 6. Nucleotide similarity (%) of partial sequences of the jhp0918 gene
The accession numbers and the aligned positions are as indicated in Table 1.
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Table 7. Nucleotide similarity (%) of partial sequences of the dupA gene
The accession numbers and the aligned positions are as indicated in Table 1.
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Fig. 2. Nucleotide sequence alignment of partial sequences of dupA from strains analysed in the current study. The asterisks refer to positions where nucleotide sequences match those of the consensus sequence and the hyphens represent deletions. The variant nucleotides are shown by shading.
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Recent studies have proposed the possibility of using genetic markers in the plasticity zone as indicators of pathogenicity for H. pylori infection, in spite of a lack of credible knowledge regarding the functions of the putatively encoded proteins in this cluster. It seems that these determinants may play a key role in determining the virulence capacity of H. pylori strains either directly or by encoding factors that may lead to varying clinical outcomes. The association between some of the ORFs in the plasticity zone and various disease categories has been previously reported. For instance, Occhialini et al. (2000) found that two single ORFs (jhp0940 and jhp0947) were more prevalent in strains isolated from patients with gastric adenocarcinoma in Costa Rica. However, Santos et al. (2003) showed the association between jhp0947 and DU as well as GC in Brazilian patients. This was once more confirmed for jhp0947 and jhp0949 genes in DU patients from the Netherlands (de Jonge et al., 2004). The implication that jhp0917 and jhp0918 promote DU and protect against GC was made by Lu et al. (2005) and the continuous gene formed by these was thus referred to as the duodenal ulcer promoting (dupA) gene. Herein, we have investigated this hypothesis in Iranian dyspeptic patients. Iran, located in South-West Asia, has a high prevalence of H. pylori infection with limited application of previously studied H. pylori virulence markers in patient screening (Talebkhan et al., 2007).
Analysis of dupA in a limited group of patients revealed that it is present in approximately half of the collected strains regardless of the clinical status. This prevalence is higher than that reported for countries such as Colombia, the United States, Belgium, India and some East Asian countries, including Japan, Korea and China, but lower than that for South Africa and Brazil. The variations in the prevalence of dupA in different countries may be partly due to distinct dyspeptic populations and geographical heterogeneity of H. pylori isolates.
In accordance with the study by Lu et al. (2005), our study confirms the presence of jhp0917 and jhp0918, linked to each other and constituting the complete dupA gene. This study also illustrated the lower distribution of non-functional strains in Iran (5.09 %) than in India (11 %) with regard to circulation of jhp0917-positive/jhp0918-negative isolates. The principal limitation of our study is the small number of DU cases, which impairs a definite conclusion. The prevalence of dupA in our limited number of DU patients was slightly higher than that in non-cardia GC, but this was not statistically significant. This is in accordance with the findings of Argent et al. (2007), who demonstrated a lack of association between the presence of dupA and DU in strains isolated from four different geographical regions, Belgium, South Africa, China and the United States. Furthermore, Gomes et al. (2007) confirmed the lack of association of dupA with DU as well as GC in both Brazilian adults and paediatric patients. In contrast to the mentioned studies, the report by Arachchi et al. (2007) confirmed the association of dupA with DU as an informative virulence marker. These discordant results may be explained by differences between the plasticity regions of H. pylori strains isolated from distinct geographical areas. On the other hand, Lu et al. (2005) reported that only a small proportion (6–12 %) of GC patients was colonized with dupA-positive strains. This was fully negated by Argent et al. (2007), who suggested a direct association between the presence of dupA and GC (due to the high prevalence of dupA in GC patients), particularly in the combined populations of Belgian and South African patients. The current findings verify the need for analysis of larger numbers of dyspeptic patients in particular DU cases.
Having studied gastric histopathological features, Lu et al. (2005) demonstrated an inverse association between possession of the dupA gene and pathological precursors of GC, including intestinal metaplasia and atrophy. Our study supports the hypothesis of this gene being a protective factor against GC, such that it was found to be inversely associated with pre-malignant histological features such as dysplasia. Despite the absence of a clear inverse association between possession of the dupA gene and GC, keeping in mind that gastric epithelial dysplasia represents the only true histological marker of GC and the increased prevalence of precursor lesions such as dysplasia as a main pre-malignant and precancerous lesion is associated with increased incidence of cancer, dupA gene may be applicable as a protective marker against GC development.
Another significant inverse relationship was found between lymphoid follicles and possession of dupA. Since lymphoid follicles represent a relatively common histological feature of chronic gastritis, which is believed to progress towards gastric malignancies, this may provide further support for dupA as a protective marker against GC.
In addition, sequencing of this gene from Iranian isolates and comparison with sequences from isolates from other geographical regions indicated more than 93 % similarity and the presence of the continuous gene due to nucleotide insertion after position 1385. The presence of a continuous gene in these isolates may represent a functional protein, which requires further investigation. In summary, we conclude that the discrepant results obtained for this putative virulence marker may be a consequence of the plasticity of H. pylori, which contributes to its genetic diversity and requires additional studies for a firm conclusion.
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ACKNOWLEDGEMENTS
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The authors would like to thank Yeganeh Talebkhan, Leili Zamaninia and Mojgan Moghadam from the HPRG (Helicobacter pylori Research Group) at the Biotechnology Research Center of the Pasteur Institute and the clinical staff at the Cancer Research Center of Tehran University of Medical Sciences and Endoscopy Unit of Amiralam Hospital for their sincere technical and clinical support. This study was funded by a generous grant from the Deputy of Research and Technology of the Iranian Ministry of Health and Medical Education in support of international research collaborations.
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Roles of the plasticity regions of Helicobacter pylori in gastroduodenal pathogenesis
J. Med. Microbiol.,
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545 - 553.
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INTRODUCTION
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