New approaches for genotyping of Helicobacter pylori based an amplification of polymorphisms in intergenic DNA regions and at the insertion site of the cag pathogenicity island

The population of the gastric pathogen Helicobacter pylori shows a high deg ree of genetic diversity. It is well established that heterogeneity at the isolate level is caused by nucleotide transitions within genes, differences in the gene order, and by genetic instability of single genes as well as o f a large virulence-associated genomic DNA region, the cag pathogenicity is land (PAI). Analysis of intergenic regions with specific PCR-assays;develop ed in this study, revealed that DNA polymorphisms in the noncoding DNA loca lized in front of the genes ribA and vacA and at the insertion site of the cag PAI contribute to the genetic diversity of H. pylori and are useful for differentiation of individual isolates. Thirteen individual genotypes were identified by PCR analysis of these polymorphic loci in 487, 241, and 182 clinical H. pylori isolates. Sequence analysis revealed that genetic variab ility in front of genes ribA and vacA, and in the intergenic region at the PAI insertion site is caused by insertion and deletions of so-far-unknown D NA sequences as well as by parts of the H. pylori IS elements IS605 and IS6 06, respectively. The new genotypes identified could be used to differentia te antrum and corpus isolates from the same patients. Their combination wit h vacA allele subtypes and with the cagA status allowed to differentiate 14 0 isolates in 51 subtypes. In 36 cases the corresponding genotype patterns were isolate specific. In summary, the results confirm that DNA polymorphis ms in intergenic regions contribute to the genetic diversity of H. pylori. Although individual H. pylori genotypes were not associated with peptic ulc er disease, the PCR-based approaches for their detection developed here sho uld be of use for further investigation of genetic diversity in H. pylori a nd for epidemiological purposes.