Association of an NFKB1 intron SNP (rs4648068) with gastric cancer patients in the Han Chinese population

Gastric cancer (GC) is the second leading cause of cancer-related deaths and the fourth most frequent cancer in the world [1]. Generally, GC mortality is ranked the highest in Eastern Asia (China, Japan), Eastern Europe, and South America compared with other cancerous tumors, whereas GC mortality is ranked the lowest in North America and most parts of Africa. The etiology of this cancer is multifactorial, involving both genetic and environmental risk factors. To clarify the genetic background of the gastric cancer, it is necessary to specifically identify the genetic factors such as single nucleotide polymorphisms (SNPs).

Nuclear factor kappa B (NF-κB) is a family of structurally related eukaryotic transcription factors that are persistently active in the pathogenesis of numerous malignancies, including prostate [2], breast [3, 4], colorectal [5], pancreatic [6], oral [7], and gastric carcinoma cancer [8]. The NF-κB family contains five members in mammals: NFKB1 encoding p50, NFKB2 encoding p52, RELA encoding p65, REL encoding c-Rel, and RELB encoding Rel-B. The most common dimer is the p65/p50 heterodimer. The human NFKB1 gene is located at chromosome 4q24. Several groups have shown that there is a relationship between gastric cancer and the polymorphisms of NFKB1 promoter regions [9, 10], and local tumor growth and lymph node spread in gastric cancer have also been proven to be associated with N-myc downstream-regulated genes in previous reports [11‐13]. This study investigated the possible association between the SNP polymorphisms of NFKB1 and susceptibility to gastric cancer and its tumor behavior.

We first screened SNPs throughout the entire NFKB1 gene locus and selected 4 SNP sites from 99 SNP sites that were in linkage disequilibrium (LD) in the Hapmap CHB (Han Chinese in Beijing, China) data: rs12509517 and rs4648068, which are in LD with the tagging SNP rs230539 (r² > 0.8), and rs4648065 and rs4648037 (r² < 0.8). The genetic associations of the SNPs were examined by comparing the genotypes of 30 unrelated gastric cancer patients with those of 30 healthy controls in a small-scale genotyping study. Then, we conducted a large-scale case–control study on the Han Chinese population to investigate the association of the rs4648068 polymorphism with gastric cancer and clinicopathologic variables of gastric cancer patients using quantitative real-time PCR (qRT-PCR).

NF-κB was reported to be a proinflammatory transcription factor, which could bind the enhancer of the kappa light chain of immunoglobulin and promote tumorigenesis [29, 30]. The rs4648068 (A > G) polymorphism was identified in a study of the analysis of variation in NFKB1 genes and expression levels of NF-κB regulated molecules, and SNP rs4648068 was subsequently reported to be associated with VCAM1 and LDL phenotypes [31, 32].

Considering there is a relationship between gastric cancer and the promoter region of NFKB1[9, 10] and many reports also showed that the genetic variations in introns might involve alternative gene regulation, transcript processing, or chromosomal rearrangements [16, 17, 23, 24], the association between the rs4648068 polymorphism in NFKB1 and gastric cancer has been studied. Here, we found that the rs4648068 polymorphism had a significantly different distribution in gastric cancer patients and healthy control individuals in a preliminary small-scale genotyping study (P = 0.029). Therefore, we investigated the role of the NFKB1 rs4648068 polymorphism in gastric cancer susceptibility in the Han Chinese population in a case–control study. The results indicated that people with the homozygous GG alleles in rs4648068 had a 2.65-fold increased risk of gastric cancer compared people carrying other rs4648068 alleles (P = 0.042).

Surprisingly, although reports showed that age and gender are the 2 major risk factors of gastric cancer, and gastric cancer is reported to be more common in men over the age of 55 than in other groups of individuals [25‐28], our stratified analysis by age and gender did not modify the association between rs4648068 and the risk of gastric cancer. Lewander’s group [33] and Gao’s group [34] have reported that the genetic polymorphisms have the opposite effect in Swedish populations compared with Chinese populations and in Asian populations compared with Caucasian populations. Whether our results are ethnically dependent requires further elucidation. In addition, although the minor allele frequency (MAF) of rs4648068 is high (0.346), this conclusion would be more convincing if it can be replicated in another cohort.

On the basis of our genetic association data in the Han Chinese population, the SNP within an NFKB1 intron region (rs4648068) showed marginal significance. This suggested that there might be correlation between NFKB1 genotypes and the gastric cancer clinicopathologic characteristics such as lymph node status and serosa invasion because of the different associations that were observed for the high-risk subsets and the low-risk subsets.

The SNP rs4648068 (A or G) resides in intron 11 of NFKB1, occupying position 103,518,305 of chromosome 4. The predominant nucleotide at this position is A for human, chimpanzee, and macaque, but G for orangutan, mouse, and dog. There is no conserved trait at this position in the genome of elephants, opossum, chicken, or zebrafish, although the corresponding region is Nfkb in these organisms. The percentages of the A and G alleles in the Han Chinese ethic group in Beijing are 0.609 and 0.391 (http://​www.​Hapmap.​org), respectively; our study in Shanghai found that the percentages of the A and G alleles are 0.551 and 0.449, respectively. The discrepancy may result from geographic factors because the percentages of A and G alleles in Han Chinese in Denver, Colorado, are also different from the above findings (A: 0.596; G: 0.404, data from http://​www.​Hapmap.​org). To elucidate the potential functional role of SNP rs4648068, we also performed a bioinformatic data mining study using the UCSC genome browser (http://​genome.​ucsc.​edu/​cgi-bin/​hgTracks) and Encode databases. The region near rs4648068 in intron 11 of NFKB1 exhibited increased levels of H3K9me1 and H3K4me1 (the highest value of H3K9me1 is 15, while the increase of the H3K4me1 level is not as remarkable as that of H3K9me1). These tracks displayed maps of chromatin state that were generated by the Broad/MGH ENCODE group using chromatin immunoprecipitation coupled with sequencing (ChIP-seq). Chemical modifications (methylation for H3K9me1 and acetylation for H3K4me1) to the histone proteins that are present in chromatin influence gene expression by changing the accessibility of the chromatin to transcription factors. The increased levels of chemo-modification imply that rs4648408 may be located in a regulatory element, such as an enhancer. Hence, the G allele of rs4648068 may alter the power of a potential enhancer, which may have an influence on disease in the host.

In the present study, we observed that the frequency of the G allele in clinical stages III + IV patients was significantly higher than that in clinical stages I + II patients, indicating that this polymorphism is associated with high clinical stage (P = 0.025). On the other hand, the stratified analysis by lymph node status showed that patients with the GG genotype and G carriers were more likely than those with the AA genotype and A carriers to have a positive lymph node status (P = 0.004 and 0.001, respectively), suggesting that this polymorphism might contribute to the constitutive NF-κB activity in gastric cancer.

Our investigations also demonstrated that the frequency of the GG homozygous_genotype in serosa invasion-positive patients was significantly higher than that in serosa invasion-negative patients (P = 0.006). Tumor invasion is regulated by numerous NF-κB target gene products, including MMP-9, TIMP-1/2, PAL 2, CXCR4, interleukin-8 (IL-8), and other chemokines [29, 35, 36]. NF-κB plays an essential role in the migration and the organ-specific homing of metastatic breast cancer cells [35, 37].

RQL designed the study and analyzed and interpreted the data. XG participated in clinical data and information collection. YC and SHL collected all samples and performed qRT-PCR genotyping experiments. YFY and JN were involved in statistical analysis of the data and drafted and edited the manuscript. LG conceived and supervised the project and reviewed the manuscript. All authors have read and approved the final manuscript.