Genome-wide association study reveals multiple nasopharyngeal carcinoma-associated loci within the HLA region at chromosome 6p21.3

Abstract

Nasopharyngeal carcinoma (NPC) is a multifactorial malignancy closely associated with genetic factors and Epstein-Barr virus infection. To identify the common genetic variants linked to NPC susceptibility, we conducted a genome-wide association study (GWAS) in 277 NPC patients and 285 healthy controls within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). Twelve statistically significant SNPs were identified and mapped to chromosome 6p21.3. Associations were replicated in two independent sets of case-control samples. Two of the most significant SNPs (rs2517713 and rs2975042; p(combined) = 3.9 x 10(-20) and 1.6 x 10(-19), respectively) were located in the HLA-A gene. Moreover, we detected significant associations between NPC and two genes: specifically, gamma aminobutyric acid b receptor 1 (GABBR1) (rs29232; p(combined) = 8.97 x 10(-17)) and HLA-F (rs3129055 and rs9258122; p(combined) = 7.36 x 10(-11) and 3.33 x 10(-10), respectively). Notably, the association of rs29232 remained significant (residual p < 5 x 10(-4)) after adjustment for age, gender, and HLA-related SNPs. Furthermore, higher GABA(B) receptor 1 expression levels can be found in the tumor cells in comparison to the adjacent epithelial cells (p < 0.001) in NPC biopsies, implying a biological role of GABBR1 in NPC carcinogenesis. To our knowledge, it is the first GWAS report of NPC showing that multiple loci (HLA-A, HLA-F, and GABBR1) within chromosome 6p21.3 are associated with NPC. Although some of these relationships may be attributed to linkage disequilibrium between the loci, the findings clearly provide a fresh direction for the study of NPC development.

Figure 1

Summary of GWAS Results, Based on Chromosome Associations with NPC were determined for 480,365 SNPs among 277 cases and 285 controls. The x axis represents the position on each chromosome from the p terminus to the q terminus, and the y axis depicts p values on a minus logarithmic scale. Analysis was adjusted for age and gender with the use of a logistic regression model. Chromosomes are shown in alternating colors for clarity. Red dots indicate SNPs with significant p values in the combined data from three stages (Table 1). Data on the X chromosome are not presented, due to gender adjustment during statistical analysis.

Figure 2

Association Analyses of SNPs at Chromosome 6p21.3 Top: Blue dots indicate the p values for association testing drawn from first-stage GWAS on a minus logarithmic scale. Red dots depict SNPs with significant p values in combined (three stages) data. Bottom: Detailed LD structure depicted in HaploView. Analysis was adjusted for age and gender with the use of a logistic regression model. The corresponding location of genes is shown below. Pairwise linkage disequilibrium estimated in 285 control samples depicted in HaploView is shown at the bottom. Increasing intensities of red represent higher r² values.

Figure 3

GABA_B Receptor 1 Protein Expression in NPC Tumor Cells GABA_B receptor 1 protein was detected in NPC tumor cells (A), infiltrating leukocytes (C, indicated by arrowheads), and endothelial cells (D, indicated by “V”) by immunohistochemical staining. Distribution of NPC tumor cells is depicted with positive nuclear EBER signals (B). Expression of GABBR1 transcripts (E) and protein (F) was detected in NPC cell lines and in the normal nasopharyngeal epithelial cell line, NP69.