Association of polymorphism of PTPN 11 encoding SHP-2 with gastric atrophy but not gastric cancer in Helicobacter pylori seropositive Chinese population

Gastric cancer is the most common malignancy of gastrointestinal tract in East Asian populations and the third most common cause of cancer-related deaths in China [1, 2]. Helicobacter pylori (H. pylori) infection has been established as a major risk factor for gastric cancer, through the induction of gastric atrophy and progression of precancerous lesions by numerous studies [3, 4]. Although H. pylori is estimated to inhabit at least half of the world’s human population, just few subjects develop to gastric precancerous lesions and adenocarcinoma. The extent of gastric damages induced by H. pylori infection seems to vary from one subject to another, suggesting that the combination of host genetic traits and bacterial virulence plays important roles in long-term outcomes of H. pylori infection [5‐7].

Several studies have provided evidences that infection with cagA-positive H. pylori associates with higher grades of gastric inflammation and is more virulent than the cagA-negative strains [8]. The CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system, where it undergoes tyrosine phosphorylation by Src and Abl kinases. Tyrosine-phosphorylated CagA then acquires capability to interact with and deregulate SHP-2 phosphatase, a bona-fide oncoprotein [9]. The formation of cagA/SHP-2 complex induces abnormal proliferation and migration of gastric epithelial cells, consequently resulting in gastric atrophy and gastric carcinoma [10‐12]. In addition, gain-of-function mutations of the SHP-2 have recently been found in human malignancies [13‐15]. Kim et al also revealed that gastric cancers displayed higher levels of SHP-2 protein compared to normal cells, suggesting that neo-expression of this signalling protein in cells might play a role in the gastric carcinogenesis [16]. Since the protein-tyrosine phosphatase nonreceptor-type 11 (PTPN11) gene encodes protein containing two tandem Src homology-2 domains, which function as phospho-tyrosine binding domains. SHP-2 closely interacts with the CagA protein, therefore, it is speculated that functional polymorphisms in the PTPN11 may mediate the interaction of this protein with its substrates and affect its regulatory role in various cell signalling events, such as mitogenic activation, metabolic control, transcription regulation, cell migration, and malignant transformation in H. pylori infected subjects.

The PTPN11 gene is on chromosome 12, containing 16 exons. Several single–nucleotide polymorphisms (SNPs) rs11066322, rs11066320 and rs2301756 have been identified in Caucasian females to be associated with apoB levels and LDL-C levels [17]. Another study demonstrated that the rs11066322 was associated with increased plasma HDL-C levels [18]. These results suggested that genetic variants influencing SHP-2 activities may modulate biological functions of the protein. In gastric cancer, Japanese group has found that a prevalent SNP in intron3 (rs2301756) was associated with an increased risk of gastric atrophy in Japanese population with H. pylori infection [19‐22]. The aim of the present study is to determine whether polymorphisms of PTPN11 gene are associated with clinical outcomes of H. pylori infection in Chinese population.

CagA-secreting H. pylori infection plays an important role in gastric carcinogenesis via a sequential CagA signal transduction pathway. CagA initially binds to seven protein components to activate aberrant cellular responses that promote the development of gastric cancer. Since the function of CagA protein is regulated by its binding partners, therefore genes that encode CagA interacting molecules may modify the risk of gastric cancer. In the present study, 5 htSNPs of the PTPN11 gene were investigated for their associations with H. pylori infection, gastric atrophy and gastric cancer in Chinese Hans population. We found that subjects bearing rs12423190 CC genotype at intron 6 had a significantly higher risk in H. pylori-seropositive gastric atrophy. The OR for gastric cancer was increased in those carrying the GCGTG haplotype versus the GTATA (OR = 1.30, 95%CI: 0.93-1.83), however, the P value was 0.09, not statistically significant. In this study, the allele frequencies of rs12423190 were 71.8% for the T allele and 28.2% for the C allele among control subjects. The frequency of the C allele was similar to that reported in the HapMap Project (31.4%). Compared to the NCBI SNP database, we found that the frequency of the C allele is obviously higher in Chinese population than other populations (23.3% in Japanese; 11.5% in Utah residents with Northern and Western European ancestry). As incidences of gastric cancer are high in Chinese, Japanese and Koreans, high frequencies of this polymorphism in Asian populations may be part of explanation. Our findings indicated that the C allele contributes to genetic predisposition to H. pylori-induced gastric atrophy in Chinese population. A further study is required to confirm associations of the PTPN11 rs12423190 polymorphism with gastric atrophy in diverse ethnic populations.

Recent studies revealed significant associations between the rs2301756 AA genotype in intron3 and reduced risk of gastric atrophy among H. pylori-seropositive Japanese subjects [7, 19, 22]. Our study found a similar trend of decreased risk of OR for rs2301756, however, the OR of atrophy was not significantly lower compared with the GA genotype (OR = 0.90, 95%CI: 0.51-1.59) and AA genotype (OR = 0.51, 95%CI: 0.06-4.09). In controversy, the association study for rs2301756 and gastric atrophy showed a completely opposite result in Uzbekistan population [20]. Zhu F and colleagues also demonstrated that the rs2301756 A allele was associated with low risk of intestinal metastasis (IM) indicating that H. pylori infection induces gastric precancerous lesions, such as gastric atrophy (OR = 0.46, 95%CI: 0.21-0.99) in H. pylori seropositive individuals. Meantime, an inverse association was shown in H. pylori-seronegative subjects in the same study (OR = 2.51, 95%CI: 1.21-4.43) [27]. There were no statistically differences in the frequency of rs2301756 in our study and others [21, 28]. Inconsistent results from different studies may be due to different environmental backgrounds and ethnic groups. In our study, gastric atrophy was only identified in 82 cases of H. pylori- seropositive subjects, the minor allele of rs2301756 was relatively rare (MAF = 0.139). The statistical power maybe not sufficient (power = 0.10) to examine the association. A further investigation of association between rs2301756 and gastric atrophy is needed in a large population and other ethnic groups.

The functions of PTPN11 polymorphisms are still unknown. SHP-2 contains two tandem SH2 domains, a PTP domain, and other functional motifs. Genetic mutations in PTPN11 exon 3(encoding the N-SH2 domain), exon 4(encoding the C-SH2 domain), Exons 7, 8 and 12(encoding the PTP domain) have been identified in Noonan syndrome, juvenile myelomonocytic leukemia, LEOPARD syndrome, lung cancer, liver cancer and colon cancer [29]. The interaction of tyrosine phosphorylated Cag-A with the SH2 domain of the protein is supposed to induce a conformational change in SHP-2 that weakens the inhibitory interaction between PTP and N-SH2 domain, and results in activation of SHP-2's catalytic activity [10, 30]. The rs12423190 polymorphism is located in the intron 6, 1408 bp upstream exon 7, encoding part of PTP domain. Using a free bioinformatic tool (http://​fastsnp.​ibms.​sinica.​edu.​tw/​pages/​inputCandidateGe​neSearch.​jsp) the rs12423190 is predicted to locate in the side of an intronic enhancer which could affect the gene transcriptional regulation. SHP-2 has several biological functions. The gain-of-function of SHP-2 may accelerate the downregulation of T-cell and B-cell activation through CTLA-4/PD-1 as well as IL-6/STAT3 signalings, eventually leading to a decrease in inflammation. Meantime, it might act as a signal promoter in inflammation. SHP-2 promotes growth factor induced activation of phosphatidylinositol 3-kinase (PI3-K)/Akt, the extracellular signal-related kinases (ERKs) and nuclear factor-kappa B (NF-κB). SHP-2 can either negatively or positively regulate the activation of Janus kinase 2 (Jak2)/signal transducer and activator of transcription (STAT) and the c-Jun-amino terminal kinases (JNKs) depending on different circumstances. rs12423190 may promote the over-expression of SHP-2, further involvement in the up-regulation of inflammatory cytokines through MAPKs and NF-κB signaling pathways, eventually leading to increase in inflammation related to atrophy [31]. However, the rs2301756 does not appear to reside in transcription factor binding sites or splicing sites, but is in the LD with associated haplotypes. The rs12423190 polymorphism is also in a linkage disequilibrium, which may contain other unidentified causative SNPs. Further studies of PTPN11 sequence variants and their biologic functions may shed light in understanding the association of PTPN11 polymorphisms and the risk of GA.

In addition, we demonstrated that gastric cancer patients had high prevalence of both H. pylori seropositivity and gastric atrophy; the prevalence of H. pylori seropositivity was significantly higher in subjects with gastric atrophy compared to controls without gastric atrophy, implying the important links with of H. pylori infection, gastric atrophy, and gastric cancer. Genetic factors, such as PTPN11 polymorphisms may contribute to gastric atrophy, through affecting the connection of Cag A and SHP-2 protein.

The first limitation in our study is the H. pylori infection status being determined by serology, not by the serum CagA antibody test. In East Asia countries, almost all H. pylori strains reported from infected patients were East Asian CagA positive strains [32‐34], therefore H. pylori strains in infected patients most likely possess CagA in our study. The second limitation is the PG criteria for GA screening, the criterion for GA is PGI <82.3 ng/ml and PGI/II ratio <6.05. These parameters for atrophy are used in China and have been validated in only one histological confirmatory study [23], it is may be insufficient to draw a reliable diagnosis, thus more confirmatory studies are needed. Finally, a small sample size of gastric atrophy, especially for the cohort of H. pylori (+) gastric atrophy individuals is due to a low incidence of gastric atrophy in China. A large-scale study for recruiting more patients with gastric atrophy is required to confirm our findings in the future.

In conclusion, our study provides the first evidence that rs12423190 polymorphism of the PTPN11 gene is significantly associated with an increased risk of gastric atrophy in H. pylori infected Chinese population, suggesting that rs12423190 polymorphism could be used as a biomarker of genetic susceptibility to gastric atrophy.