Nasopharyngeal carcinoma (NPC), a malignancy that forms in the epithelium of the nasopharynx, has a distinct geographic distribution and is highly prevalent in southern China, Southeast Asia, and North Africa. Although all Han Chinese populations exhibit an increased risk of NPC, the incidence rate varies by region. For example, male populations in Guangdong and Guangxi in southern China have consistently exhibited a higher incidence rate (20.6–39.94/100 000 person-years) compared with those in moderate-incidence regions, such as Taiwan (8.6/100 000 person-years), and those in most of the Western world (less than 1/100 000 person-years) [1–5]. The etiology of NPC is multifactorial, involving genetic components, Epstein–Barr virus infection, and other types of environmental exposure [1]. The variations in NPC incidence might be due to differences in environmental exposure among geographic regions; however, the genetic components underlying the differences in incidence in Han Chinese populations remain underexplored.

The genetic association of human leukocyte antigen (HLA) class I genes, particularly HLA-A, with NPC was established in 1974 [6] and has been confirmed in more than 100 association studies adopting traditional HLA genotyping techniques. Studies have consistently identified an association between NPC and HLA-A*1101, HLA-A*0207, and HLA-B*5801 [7–9]. The distribution of these three alleles in the human genome appears to be consistent with the geographical distribution of NPC incidence in southeastern China; the allele frequency is particularly high in regions with high NPC incidence rates [10–12]. However, no difference in HLA allele frequency has been observed in the results of NPC association studies conducted in regions with various incidence rates [13–15], suggesting that the HLA genes might not directly lead to differences in NPC incidence.

Three independent genome-wide association studies (GWASs) [16–18] have identified multiple significant association signals in chromosome 6p21.3 near the HLA-A gene, which exhibited extremely strong linkage disequilibrium (LD) (Fig. 1). These observations raised the question as to whether these associated single-nucleotide polymorphisms (SNPs) represent an independent effect or are only proxies of the HLA-A gene. Studies conducted in medium- and high-incidence regions (Taiwan [16, 19], and Guangdong and Guangxi [17, 18], respectively) have yielded contradictory conclusions.

To evaluate the effect of HLA-A and its neighboring gene on NPC susceptibility, we conducted a meta-analysis on the association between the five most frequently studied chromosome 6p21.3 SNPs (rs9260734, rs2517713, rs3129055, rs29232, and rs29230) and NPC susceptibility. Thus far, no meta-analysis has been conducted to explore the overall NPC risk and the genetic heterogeneity associated with chromosome 6p21 SNPs. In the current study, we postulated that moderate heterogeneity in rs29232 might contribute to the regional variation in NPC incidence rates.

In the present meta-analysis, four of the five chromosome 6p21.3 polymorphisms exhibited strong and consistent positive associations with NPC. The three SNPs (rs2517713, rs9260734, and rs29230) yielded highly consistent results with no heterogeneity, despite differences in the genotyping platform (Table 1) and the use of surrogate SNPs for rs2517713 (Table 2). The strongest association was discovered in rs2517713 near the HLA-A gene (P = 1.08 × 10⁻⁹¹, OR = 0.58, 95 % CI = 0.55–0.61), further confirming the critical role of the HLA-A gene in the susceptibility of NPC.

NPC has a distinct ethnic and geographic distribution. The allele frequencies of NPC-associated alleles (HLA-A*1101, HLA-A*0207, and HLA-B*5801) in the human genome are consistent with the geographical distribution of NPC incidence in southeastern Asia [10–12], suggesting that the genetic differences among populations might play a crucial role in NPC incidence. However, in the NPC association studies, the frequency of HLA alleles did not differ noticeably in populations with dissimilar incidence rates, suggesting that HLA genes might not directly cause this difference. In the current meta-analysis, we did not observe heterogeneity in the HLA-A SNPs, which supports our hypothesis. By contrast, our analysis indicated that rs29232 exhibited distinct features in Han Chinese populations in regions with different incidence rates. First, the heterogeneity of rs29232 was markedly reduced when we stratified the meta-analysis according to the incidence region. Second, in the subgroup analysis, the OR of rs29232 was higher in moderate-incidence regions than in the high-incidence regions. Finally, in previous NPC association studies, rs29232 was independent of the HLA-A effect in moderate-incidence regions, but not in high-incidence regions. These results suggest that rs29232 might contribute to the difference in NPC incidence in Han Chinese populations.

Studies conducted in regions with different incidence rates have yielded inconsistent results regarding rs29232. A GWAS conducted in Taiwan, a moderate-NPC-incidence region, using multiple logistic regression analysis and stepwise logistic regression concluded that rs29232 was significantly associated with NPC, even after the removal of the HLA-A SNP (rs2517713) and sequence-based HLA-A alleles (HLA-A*0207/0215 N and HLA-A*110101/0121 N) [16]. Another independent post-GWAS case–control study conducted in Taiwan yielded similar results, indicating that HLA-A and rs29232 are likely to be independent risk factors for NPC, and that NPC risk is highest among people carrying homozygous HLA-A*0207 and rs29232 risk alleles [19]. By contrast, a GWAS conducted in Guangdong, a high-NPC-incidence region, revealed that the strength of the association with rs29232 greatly diminished after the researchers controlled for the effect of rs2860580 (HLA-A), whereas the strength of the association with rs2860580 (HLA-A) decreased after they adjusted for rs29232. Collectively, these results suggest that the associations with rs29232 and rs2860580 (HLA-A) were correlated rather than probabilistically independent [17]. In an NPC GWAS conducted in Guangdong and Guangxi, a similar multivariate logistic regression analysis indicated that rs29232- and GABBR1-related SNPs were nonsignificant after adjustment for HLA-A-related SNPs and alleles. The authors of the study stated that all the other significant associations identified were only proxies for HLA-A*1101 because of the strong LD within the region [18]. To summarize, study findings from moderate-incidence regions support the independent role of rs29232, whereas those from high-incidence regions indicate that only one true association signal (HLA-A) exists within this chromosome region.

In the current study, no heterogeneity was observed among studies except in rs3129055 (I² = 58 %, P = 0.0361, HLA-F) and rs29232 (I² = 47 %, P = 0.1092, GABBR1). However, the heterogeneity in rs29232 was not statistically significant (P > 0.1). Because tests of heterogeneity are severely underpowered in analyses of only a few studies [33], heterogeneity might still exist despite a lack of statistical significance. Since the meta-analysis result for rs3129055 did not achieve genome-wide significance (<10⁻⁷), we excluded it from further analysis. The moderate level of heterogeneity (I² = 47) of rs29232 markedly decreased (I² = 0) when we stratified the study population according to geographic region (Table 3), suggesting that regional differences in NPC incidence caused the heterogeneity. Furthermore, the ORs were higher in the moderate-incidence regions (OR = 1.70, 95 % CI = 1.47–1.95) than in the high-incidence regions (OR = 1.40, 95 % CI = 1.30–1.49). Since rs29232 was a significant association signal independent of the effect of the HLA-A gene in the moderate-incidence regions, we suspected that rs29232 caused the regional difference in NPC incidence among Han Chinese people. Compared with high-incidence populations, moderate-incidence populations might be more strongly affected by rs29232.

Although the current evidence on the role of rs29232 and the GABBR1 gene seems self-contradictory, previous studies have provided consistent findings regarding the role of GABBR1 in NPC. GABBR1 encodes the protein gamma-aminobutyric acid B receptor 1 (GABBR1), a G protein-coupled receptor that forms a heterodimer with GABAB receptor 2, thereby triggering downstream signaling events involved in the proliferation, differentiation, and migration of cancer cells. One study reported that the intensity of the GABBR1 signal in tumor cells was significantly higher than that detected in adjacent normal epithelial cells (P < 0.001) in the immunohistochemical staining of NPC tissues [16].

The rs29232 SNP exhibited a lower LD (r ²  < 0.6) than did all other SNPs in the HapMap CHB data set in the MHC region. This finding suggests that the role of rs29232 might not directly relate to the downstream GABBR1 gene or other neighboring genes, but rather affect genes located in other regions or chromosomes through long-range cis-acting or trans-acting. Nevertheless a recent genome-wide SNP–SNP interaction analysis detected no significant interaction between rs29232 and other SNPs [34]. Further investigation of the role of rs29232 and its relationship with the incidence and etiology of NPC is vital. In explorations of the genetic factors underlying disease susceptibility, epidemiological factors, such as disease incidence rates, are rarely considered. In the current study, an association analysis of rs29232 in regions with high or moderate incidence rates was performed. Future studies conducted in low-incidence regions such as northern China may clarify the relationship between genetic factors and geographic incidence rates. In addition, using imputation or next-generation sequencing to explore the detailed genotypes near rs29232 can further reveal the genetic causes underlying the variation in regional NPC incidence rates. The current study suggested that genetic effects might be involved in epidemiological factors such as regional disease incidences. However, epidemiological factors have rarely been considered in large cross-region association studies. Thus, we suggest that future large cross-region meta-analyses include geographic incidence rates as potential confounding factors.

In this meta-analysis, the results indicated a strong effect of HLA-A on NPC susceptibility and a potential role of rs29232 in the regional differences in NPC incidence among Han Chinese people. However, this study had several limitations. First, although the conclusion was based on several previous studies, the heterogeneity for rs29232 observed in this study was nonsignificant. Second, although the number of subjects in the studies included in the analysis was high, the number of studies included was relatively low. Finally, meta-analyses constitute retrospective research and, thus, are subject to methodological limitations. To minimize potential bias, we used standard methods for study selection, data extraction, and data analysis. Nonetheless, the results presented here should be interpreted with caution until additional studies on rs29232 that control for incidence rates according to region are conducted.