In this study, we assessed the associations of the functional VNTR downstream of the TERT gene MNS16A with the risk of occurrence and progression of NPC in the Guangxi population in southern China. No genetic association was found between this polymorphism and the progression of NPC. However, this polymorphism was significantly associated with the onset of NPC, especially in poorly differentiated SCC. This is the first report, to our knowledge, of the genetic association between the TERT MNS16A and risk of NPC, confirming the initial hypothesis that the TERT may play a role in the pathogenesis of this malignancy.
In our study population, the S allele (SL + SS genotype) was over-represented in controls compared with cases. Furthermore, carriers of the S alleles tended to have an older age of diagnosis than those of the LL genotype, suggesting a protective role of the MNS16A S allele against the onset of NPC. It was reported that the region containing MNS16A had some promoter activity that was influenced by the length of the MNS16A tandem-repeats polymorphism . The MNS16A S allele, compared with the L allele, was associated with higher expression of the TERT antisense RNA transcripts. In our study, we further investigated the protein expression of TERT in NPC and non-cancerous nasopharyngeal epithelium tissues by IHC. We found that the SL genotype was significantly associated with lower TERT expression than the LL genotype (Table 4). Given the role of TERT in the development of NPC, it might be expected that people who carry the MNS16A S allele, and thus have more antisense TERT RNA and lower TERT protein expression (and thereby lower telomerase activity) over a lifetime, would have a lower susceptibility to developing this disorder. However, the exact functional molecular mechanisms of MNS16A remain unknown. Further studies will be needed to clarify how MNS16A affects antisense RNA transcription and how antisense RNA affects TERT expression.
Recently, the TERT MNS16A has been studied in many cancers, but the results are conflicting. Consistent with this study in NPC, one study reported an association of the MNS16A S allele with a decreased risk of non-small-cell lung cancer (NSCLC) . By contrast, a very recent study found no association between MNS16A and risk of glioma, glioblastoma and meningioma , and some reports have shown a reverse association with the risk allele, with the MNS16A S allele conferring an increased risk of breast cancer, glioblastoma and anaplastic gliomas [24, 26]. Recently, some reports have questioned the validity of the S and L combining system, and assessed directly the association of the raw alleles with the diseases. For example, Jin et al. found that the 243 bp allele, but not the 272 bp allele, was associated with the risk of lung cancer in a Korean population . Hofer et al. reported that only the 274 bp allele was associated with the risk of colorectal cancer . In this study we found that both the 243 bp and 272 bp alleles were not associated with the risk of NPC. These conflicting results could be attributable to the different ethnicities of study populations and/or different tumorigenesis of different cancers. Additionally, other factors in the studies, such as small sample size or inadequate adjustment for confounding factors, could also cause the inconsistent results. Consequently, additional well-designed case-control studies on a wide spectrum of cancers with ethnically diverse populations are warranted to understand the roles of the MNS16A polymorphism in the etiology of cancers.
We compared the genotype frequencies of the MNS16A polymorphism in populations from other studies based on available published data to identify differences from our own study. We found that the frequencies of the SS, SL and LL genotype in our controls were 0. 2%, 8. 9% and 90. 9% (Table 1), respectively, similar to those of a Nanjing population of Chinese descent (0. 4%, 9. 8% and 89. 8% in controls, respectively)  and a Korean population (0. 2%, 10. 7% and 89. 1% in controls, respectively)  described previously. We also found that the frequencies of the MNS16A genotypes were similar for controls of Caucasian descent from America, France, Austria, the UK and Nordic countries, with the SS, SL and LL genotype frequencies being 9. 2 to 13. 9%, 40. 3 to 47. 2% and 43. 6 to 47. 8%, respectively [21, 26–28]. Obviously, the frequencies of the MNS16A genotypes were significantly different from those of populations of Chinese descent (P < 0. 05, χ2 test). It remains to be determined whether these differences between ethnic groups influence the risk of developing NPC.
Previous studies have also reported that the TERT MNS16A polymorphism was associated with clinical progression in some types of cancer, including clinical stage, tumor stage, lymph-node metastasis and survival time, although the results were contradictory. Wang et al. observed a significantly prolonged survival time for patients with glioblastoma with the SS genotype compared with the SL or LL genotype . However, this finding contradicted those from other studies, in which the LL genotype was significantly associated with longer survival time in patients with glioblastoma  and early-stage (I or II) NSCLC , and was associated with decreased risk of axillary lymph-node metastasis in patients with breast cancer . It is also discrepant with the study by Carpentier et al., which did not find any survival differences for patients with glioblastoma or anaplastic glioma with different MNS16A genotypes . In patients with NPC, increased telomerase activity and TERT expression was seen more frequently in patients with advanced clinical stage (III to IV) and lymph-node metastasis (N1 to N3) [18, 19]. However, in the present study, we did not find any significant associations between the MNS16A polymorphism and the pathological stages of NPC (Table 3). These findings may be due to the different molecular mechanisms of carcinogenesis in different cancers and/or to the different ethnicities of study populations. In addition, these findings could be the result of the limited sample size and statistical power of our study. For example, this study has only a power of 28. 1% (I + II vs. III + IV), 21. 7% (T1 + T2 vs. T3 + T4), 22. 0% (N0 + N1 vs. N2 + N3) and 3. 8% (M0 vs. M1) to detect an OR of 0. 70 for carriers of the SL + SS genotypes relative to the carriers of the LL genotype. Therefore, we urge that the role of the MNS16A in the progression of NPC be investigated in additional studies with larger sample sizes.
Recently, numerous genome-wide association studies (GWAS) have been carried out to identify associations between cancer risk and single-nucleotide polymorphisms (SNPs). Multiple independent GWAS have all shown the TERT-CLPTM1L locus on 5p15. 33 to be significantly associated with many different types of cancer, including basal cell carcinoma, glioma, lung, prostate, bladder, testicular germ cell, pancreatic, and cervical cancers . Thus, the evidence that the TERT locus contains risk factors for cancer is compelling. Further studies are needed to systematically verify the SNPs at the TERT locus, investigate the interactions between these SNPs and MNS16A, and and assess their contributions to the endemics of NPC in the Chinese population.
In reviewing the results of this study, several potential limitations should be kept in mind. First, as this was a hospital-based study, our NPC cases were enroled from the hospitals, and the controls were selected from the community population, thus inherent selection bias cannot be completely excluded. However, through further adjustment and stratification in data analyses, we aimed to minimize the potential confounding effect. Second, several association studies have reported identification of the genes that may relate to the susceptibility to NPC [35–39]; however, most of the results could not be replicated in subsequent studies in other populations. Although we found a significant association between MNS16A and risk of NPC, our initial findings should be independently verified in other populations with a high incidence rate of NPC, such as other southern Chinese, Taiwanese and Singaporeans. Without rigorous replication we cannot exclude the possibility that these findings are due only to chance. Therefore, any association reported in the present study should be interpreted with great caution.