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The study aimed to investigate the relationship between gastroduodenal disease and the diversity of the cagA 3′ variable region and the amino acid polymorphisms in the Glu-Pro-Ile-Tyr-Ala (EPIYA) segments of the CagA C-terminal region of Helicobacter pylori (H. pylori).Gastric mucosal specimens from 170 patients in our center (Nanjing, Jiangsu Province, China) were collected and the genomic DNA of the H. pylori strains was extracted directly from biopsied specimens. Polymerase chain reaction (PCR) was used to amplify the cagA gene, and diversity in its 3′ variable region was assessed by direct sequencing.A total of 154 (90.6%) H. pylori isolates were cagA-positive, but the presence of this gene alone was not associated with the type of gastroduodenal disease. A total of 151 (88.8%) strains had the East Asian type EPIYA-D sequence, most of which were of ABD subtype. Three isolates from patients with chronic gastritis possessed the EPIYA-C segment. The sequences flanking the EPIYA motifs contained polymorphisms at seven residues, among which amino acid positions 878 and 879 had a statistically significant association with gastric cancer (P = 0.021). Amino acid change from glycine to aspartic acid at residue 968 was present only in patients with gastric cancer (4/20) (P < 0.001).Most H. pylori strains present in our study are of the CagA-ABD subtype. Polymorphisms at amino acids 878 and 879 flanking the EPIYA-A motif are statistically associated with gastric cancer.