In the present study, we firstly investigated the RASSF1A A133S polymorphism and the risk to ESCC and GCA development on the patients from Linzhou, the high incidence area for both ESCC and GCA. Our results demonstrated that the existence of polymorphic allele of RASSF1A might participate in gastric cardia carcinogenesis. The carriers with Ser/Ser genotype (the homozygote for codon RASSF1A A133S) and with mutated T allele genotype (Ala/Ser+Ser/Ser genotype) could have 9-fold (OR=9.01, 95% CI=0.99–83.31) and 2-fold (OR=2.06, 95% CI=1.09–3.97) higher risk to GCA, respectively. In contrast, the presence of SNP A133S in RASSF1A seems to be not involved in esophageal squamous cell carcinogenesis (P=0.69). Histopathologically, the predominant type for gastric cardia is adenocarcinoma, but the predominant type for esophageal cancer is squmous cell carcinoma in Chinese population, which may partially explain the genetic risk difference observed in the present study. It has been reported that single nucleotide polymorphism at codon 133 of the RASSF1 gene is preferentially associated with human lung adenocarcinoma risk, but not for human lung squamous cell carcinoma .
In this study, we also investigated RASSF1A promoter methylation in ESCC and GCA. Aberrant RASSF1A promoter methylation was found in 53% (76/143) of Chinese ESCC, significantly increased 5.9-fold higher the risk to ESCC (OR=5.90, 95% CI=2.78–12.52). It was apparently higher than in Korean ESCC (14%, 7/50)  and Japanese ESCC (24%, 13/55) . Interestingly, another two reports also show higher frequency of RASSF1A promoter methylation in Chinese ESCC from Hong Kong (34%, 22/64)  and Hangzhou (48.5%, 32/66), the high incidence area for ESCC in China. It is noteworthy that Chinese ESCC from low incidence area seems to have low frequency of RASSF1A promoter methylation (14.9% 22/64) . Obviously, the RASSF1A promoter methylation may be interfered by genetic and environmental factors from different population. In this study, we found that the prevalence of RASSF1A promoter methylation and the risk to ESCC in ESCC patients with the age of more than 50 years old were higher than those with less than 50 years old (OR=3.11, 95% CI=1.10–8.73), which further indicate the impact of environmental factor in terms of exposure time on RASSF1A promoter methylation.
Of particular interest, we also demonstrated the similar hypermethylation of RASSF1A in GCA (65%, 60/92), the promoter hypermethylation of RASSF1A significantly increased 7.5-fold higher the risk to GCA (OR=7.5, 95% CI=2.78–20.23), which is very similar as in ESCC. It is noteworthy that these GCA patients enrolled in our study were from the same high incidence area as the ESCC patients, suggesting that there may be similar environmental carcinogenic factors involved in ESCC and GCA. GCA seems to occur together with ESCC in China and bears many similarities in terms of concurrent geographic distribution and environmental risk factors including nutritional deficiencies, low intake of vegetables and fruit, and low socioeconomic status . Recent GWAS studies have demonstrated the similar genetic changes for ESCC and GCA in Chinese population . Furthermore, our results is close to the findings of RASSF1A gene methylation in GCA patients from Hebei province (58.7%, 54/92) , another high incidence area for ESCC and GCA in TaiHang Mountain.
It is noteworthy that, in the present study, we also observed the RASSF1A promoter methylation in the normal esophageal and gastric cardia epithelial tissue adjacent to corresponding ESCC and GCA (in ESCC: 13%, 23/143; in GCA: 20%, 19/92). These histological normal tissue has been exposed to same carcinogenic factors with tumor tissues. The present results indicate that RASSF1A promoter methylation may be a promising early indicator for esophageal and gastric cardia carcinogenesis. Further studies are required to characterize the RASSF1A promoter methylation in esophageal and gastric cardia precancerous lesion.
Another interesting finding in this study is that RASSF1A promoter hypermethylation could significantly downregulated RASSF1A protein expression both in ESCC and GCA, further indicating the possible crucial role of RASSF1A promoter hypermethylation and protein expression in esophageal and gastric cardia carcinogenesis.
Finally, considering the obvious impact of environmental factors on DNA methylation, the advantage of our study is the large sample size with similar lifestyle background. All the ESCC and GCA patients and control subjects enrolled in this study were from the same high incidence area for both ESCC and GCA.