Molecular epidemiologic studies on helicobacter pylori infection and stomach cancer risk

Helicobacter pylori (H. pylori) infection increases stomach cancer risk. The aim of this thesis was to study genetic susceptibility from the host and to develop molecular methods for future characterization of bacterial virulence factors in longitudinal cohorts.

In Study I, we investigated the association between genetic variation in an O-glycan transferase encoding gene (a4GnT) and H. pylori infection and gastric cancer risk in a Polish population-based case–control study (273 gastric cancer patients and 377 controls). A haplotype at rs2622694–rs397266 was associated with H. pylori infection, with the A-A haplotype associated with a higher risk compared with the most frequent G-G haplotype (odds ratio 2.30; 95% confidence intervals 1.35– 3.92). Neither this haplotype nor the tagSNPs were associated with overall gastric cancer risk.

In Study II, we characterized genomic evolution of H. pylori over 20 years in the stomach. Whole genome of 21 sequential isolates 20 years apart, from 7 patients, were sequenced using 454 sequencing platform. There were on average 260 point mutations (range 70 to 488) per isolate over 20 years, and 45 recombinations (range 18 to 92). Genes in the cell motility category were overrepresented in point mutations and recombinations. Specifically, mutations often affected genes involved in chemotaxis, vacuolating cytotoxin-like protein, restriction and type IV secretory pathway; and recombinations affected glycosyltransferase involved in lipopolysaccharide biosynthesis. The major form of single nucleotide substitutions was transition (85%) and the minor form was transversion (15%). Mutation was sequence context-dependent.

Clinical samples are often precious and of trace amounts. In Study III, we developed novel methods for DNA shotgun library construction and quantification. As compared with the standard procedure, our double-stranded and Y library construction methods are simpler and more efficient. A highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) was developed to quantify the amount of effective library. We also demonstrated that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labor intensive and costly titration can be eliminated and trace amounts of starting material is applicable.

Archived formalin-fixed and paraffin-embedded (FFPE) biopsies, coupled with long term follow-up, are valuable resources for molecular epidemiologic studies. Study IV presented a method based on laser capture micro-dissection and modified whole genome sequencing methods to obtain metagenomic profiles of H. pylori from 15-year old FFPE biopsy sections.

List of scientific papers

I. Zheng Z, Jia Y, Hou L, Persson C, Yeager M, Lissowska J, Chanock SJ, Blaser M, Chow WH, Ye W. Genetic variation in a4GnT in relation to Helicobacter pylori serology and gastric cancer risk. Helicobacter. 2009 Oct;14(5):120-5.
https://doi.org/10.1111/j.1523-5378.2009.00708.x

II. Zongli Zheng, Kerstin Stake-Nilsson, Anders F. Andersson, Rolf Hultcrantz, Weimin Ye, Lars Engstrand. Genomic evolution of Helicobacter pylori in human stomach over twenty years. [Manuscript]

III. Zheng Z, Advani A, Melefors Ö, Glavas S, Nordström H, Ye W, Engstrand L, Andersson AF. Titration-free massively parallel pyrosequencing using trace amounts of starting material. Nucleic Acids Res. 2010 Jul;38(13):e137.
https://doi.org/10.1093/nar/gkq332

IV. Zongli Zheng, Anders F. Andersson, Weimin Ye, Olof Nyrén, Staffan Normark, Lars Engstrand. A method for metagenomics of Helicobacter pylori from archived formalin-fixed gastric biopsies permitting longitudinal studies of carcinogenic risk. [Manuscript]