Common Genetic Variations in the MUC5AC Gene are Not Related to Helicobacter pylori Serologic Status

whether or not this association was mediated through H. pylori infection elucidated. Abstract Several lines of evidence suggest that MUC5AC genetic polymorphisms might confer susceptibility to H. pylori infection and therefore gastric cancer risk. We here assessed the association of common polymorphisms in the MUC5AC gene with H. pylori seroprevalence using an LD-based tagSNP approach in a north-western Chinese Han population. A total of 12 tagSNPs were successfully genotyped among 281 unrelated ethnic Han Chinese who had no cancer history , and no identifiable gastric disease or genetic disease. No significant association between any alleles , genotypes or haplotypes and H. pylori seroprevalence was observed. Our results suggest that common genetic variations in MUC5AC gene might not make a major contribution to the risk of H. pylori infection. study , we evaluated the associations between MUC5AC common polymorphisms and risk of H. pylori infection using tagSNP approach in the same population.


Introduction
Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic, flagellated bacterium that colonizes the human stomach (Osman et al., 2014). It is the major cause of chronic active gastritis, gastric and duodenal ulcers, atrophic gastritis, gastric cancer, and gastric mucosaassociated lymphoid tissue lymphoma (Lillehoj et al., 2012). Studies have shown that host genetic factors are estimated to contribute 57% of variation in acquisition of H. pylori infection (Malaty et al., 1998). And host genetic variations can influence the susceptibility to H. pylori infection (Zheng et al., 2009;Mayerle et al., 2013).
Upon infection, H. pylori primarily reside within the mucus layer, adhering to mucins, high molecular weight glycoproteins and major components of the protective layer across the upper mucous surfaces (Peek et al., 2002). Normal gastric mucosa shows cell type specific expression of secreted mucin MUC5AC in the surface epithelium (Wang et al., 2006;Lindén et al., 2010). Several studies have strongly suggested that MUC5AC forms the major receptor for H. pylori in the human stomach (Van de Bovenkamp et al., 2003;Lindén et al., 2008), and the infection of H. pylori can alter the expression of MUC5AC (Kocer et al., 2004).
Our previous study suggested that common polymorphisms in MUC5AC gene were associated with the risk of non-cardia gastric cancer risk in north-western Chinese Han population (Zhou et al., 2014). However, whether or not this association was mediated through H. pylori infection has not been elucidated. So in this study, we evaluated the associations between MUC5AC common polymorphisms and risk of H. pylori infection using tagSNP approach in the same population.

Study population
A case-control study on non-cardia gastric cancer was conducted in Baotou, inner Mongolian Autonomous Region of north-western China between June 2008 and December 2010, as described previously (Zhou et al., 2014). Cases were patients newly diagnosed with histologically confirmed non-cardia gastric cancer. Controls were randomly selected from a community health examination program and frequency matched to the cases by age (± 5 years) and sex. The subjects of present study were the normal controls from the case-control study. Briefly, a total of 281 unrelated ethnic Han Chinese (220 males and 61 females, mean age: 59.10±11.57 years) were included in this study. All subjects had no cancer history, and no identifiable gastric disease or genetic disease. At recruitment, informed consent was obtained from each subject, and the study was approved by the institutional review board of Baotou Medical College.

Tests for H. pylori serologic status
The serologic status of H. pylori was determined by commercial enzyme-linked immunosorbent assay (ELISA) kits (Biohit, Helsinki, Finland). As an indicator for current or previous infection, seroprevalence was defined as an anti-H. pylori IgG titer equal to or greater than 30 EIU, according to the manufacturer's recommendation.

TagSNP selection and genotyping
The selection and genotyping of tagSNPs were previously described by Zhou et al. (Zhou et al., 2014). Briefly, According to HapMap Phase 2 information for Chinese Han population (http: //hapmap.ncbi.nlm.nih. gov), 14 SNPs with a pairwise r2 ≥0.8 and minor allele frequency (MAF) ≥0.05, were selected using Tagger algorithm as implemented in Haploview. Genomic DNA was extracted from leucocytes of peripheral blood by proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation. TagSNPs were genotyped by the TaqMan allelic discrimination according to manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). And the genotyping was performed at Chinese national human genome center, Beijing. Twelve SNPs were successfully genotyped. Data from Zhou's study were used for the present analysis.

Statistical analysis
All statistical analyses were conducted by SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA).
Hardy-Weinberg equilibrium was tested by a goodnessof-fit χ2 test. Haplotypes were constructed based on the LD blocks derived from the Haploview 4.0 program. Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) adjusted for age and sex for H. pylori seroprevalence with alleles, genotypes and haplotypes.

Results
The genotype frequencies of all SNPs followed Hardy-Weinberg equilibrium in the subjects. Allele and genotype frequencies as well as ORs and 95%CI for the selected SNPs are shown in Table 1 and Table 2 Sum of column did not add up to total study subjects because of missing data; b Adjusted for age and sex  Sum of column did not add up to total study subjects because of missing data; b Adjusted for age and sex  DOI:http://dx.doi.org/10.7314/APJCP.2014.15.24.10719 Common Genetic Variations in the MUC5AC Gene in Relation to Helicobacter pylori Serologic Status significant association between any alleles or genotypes and H. pylori seroprevalence was observed.
Based on the LD data in our study, 12 tagSNPs formed 2 blocks and several singletons. Similarly as for singlelocus analysis, none of the haplotypes were associated with H. pylori seroprevalence (Table 3).

Discussion
Chronic inflammation of the gastric mucosa resulted from H. pylori infection is a serious public health problem worldwide. To date, accumulating data has showed that host genetic factors contribute to the susceptibility to H. pylori infection. For example, there are differences in H. pylori susceptibility between African Americans and US residents of European ancestry after adjusting for socioeconomic status, age and living conditions (Graham et al., 1991). Approximately 5% to 10% of a population is never infected with H. pylori, even in the presence of high exposure rates (Bardhan, 1997). Twin study has showed that there is significantly higher concordance for H. pylori infection in monozygotic compared with dizygotic twins, with a heritability estimate in twins of 57% (Malaty et al., 1994). A genome-wide association study suggests that host genetic factors confer susceptibility to H. pylori infection (Mayerle et al., 2013). Some polymorphisms are associated with the increased risk of H. pylori infection (Zhao et al., 2012;Cao et al., 2013).
H. pylori colonization of the stomach is initiated through pathogen binding to cell surface receptors expressing the sialyl-Lewis a (sLea), Lewis b (Leb), and sialyl-Lewis x (sLex) glycoconjugates (Lindén et al., 2008). The corresponding H. pylori adhesions, blood group antigen binding adhesion (BabA) and sialic acid binding adhesion (SabA), interact with these host receptors (Lindén et al., 2008). Among the epithelial glycoproteins containing these Lewis antigens are gastric mucins. MUC5AC is a mucin core protein of the gastric surface mucous cells and a major mucin core protein of the mucins forming the gastric surface mucous gel layer covering the gastric mucosa (Ho et al., 2004). Either at low pH or under neutral pH conditions, BabA can binds to the MUC5AC mucin glycoprotein (Lillehoj et al., 2012). Upon H. pylori infection, the expression of MUC5AC has altered (Kocer et al., 2004). Furthermore, MUC5AC genetic polymorphisms are associated with the risk of gastric cancer, a H. pylori related carcinoma (Jia et al., 2010;Zhou et al., 2014). Given the importance and the potential biological mechanism of MUC5AC, it is conceivable that the genetic variation in MUC5AC gene may potentially play an important role in the development of H. pylori infection. So in this study, we investigated the association between MUC5AC common polymorphisms and H. pylori susceptibility using tagSNP approach. However, our results indicated that no significant association existed between tagSNPs of MUC5AC gene and H. pylori seroprevalence.
To date, only one population-based case-control study has been conducted to investigate the association between polymorphisms in MUC5AC gene and H. pylori infection in Polish population, and found no significant association existed (Jia et al., 2010). More interesting, the study also found that common genetic variations had an effect on the risk of non-cardia gastric cancer, which is consistent with our study, suggesting MUC5AC polymorphisms might be involved in other processes besides bacterial binding in developing gastric cancer. Further studies are needed to elucidate the underlying mechanisms.
Because the loss of H. pylori from the stomach and reduced immune response often occurs during gastric carcinogenesis (Farinati et al., 1993), it is difficult to measure H. pylori infection in patients with gastric cancer. Furthermore, gastric cancer cases have probably received long-term H. pylori eradication therapy, and significant serological changes would possibly occur over the long term of H. pylori eradication therapy. So as other studies (Zheng et al., 2009;Jia et al., 2010), we did not evaluate the correlation between MUC5AC polymorphisms and H. pylori infection in patients with non-cardia gastric cancer. A prospective study should be further conducted to analyze the relationship between genetic polymorphisms in MUC5AC gene and susceptibility to H. pylori infection in patients with non-cardia gastric cancer.
In conclusion, this preliminary study suggests there is no significant association between common genetic variations in MUC5AC gene and H. pylori seroprevalence. This finding requires replication in other larger studies.