Sequence Variants At The TERT-CLPTM1L Locus On 5p15.33 Associate With Nasopharyngeal Carcinoma

Background & Aim:Sequence variants at the TERT-CLPTM1L locus have been reported to be associated with many cancers. Given the function of TERT (telomerase reverse transcriptas) and CLPTM1L (cleft lip and palate transmembrane 1 like) gene in cancer, this region is an attractive candidate susceptibility locus of nasopharyngeal carcinoma (NPC). In this study we assessed the genetic association of sequence variants at the TERT-CLPTM1L locus with the risk of NPC.Methods:The haplotype-tagging SNPs (htSNPs) were selected from TERT-CLPTM1L locus by bioinformatics analysis and literature's investigation. All the htSNPs were firstly genotyped in a cases-controls population recruited from Guangxi province (855 patients with NPC and 1036 controls without cancer). The genetic associations with the risk and severity of NPC were analyzed by logistic regression. The main genotyping methods are Sequenom iPLEX Gold technology and Genome-Lab SNPstream 12-plex genotyping platform. The SNPs, which were associated with susceptibility to NPC in Guangxi case-control population, were validated in the other independent populations, including a case-control population recruited from Guangdong province (997 patients with NPC and 972 controls without cancer) and a nuclear families population recruited from Guangxi province (231 families). Functional prediction of the SNPs which were associated with the susceptibility to NPC in multiple independent populations was performed by bioinformatics analyses. Dual-luciferase reporter assaies were performed to test the difference of the enhancer activity between two alleles of the SNPs. We also compared the length of telomere, TERT mRNA and protein levels, CLPTM1L mRNA and protein levels between the different genotypes of the SNPs.In addition, we assessed the association of MNS16A, a functional tandem repeats polymorphism in the downstream region of the TERT gene locus, and risk of NPC in the case-control population recruited from Guangxi province. This polymorphism was genotyped by PCR and electrophoresis. The genetic associations with the occurrence and progression of NPC were analyzed by logistic regression.Results:During the discovery stage,26 htSNPs across the TERT-CLPTM1L region were selected and genotyped in the Guangxi NPC case-control population. After multiple testing correction, four SNPs (rs2736098, rs2735845, rs401710 and rs401681) were significantly associated with the NPC risk after adjustment for age, sex, smoking status, alcohol use and family history. Replications of these four SNPs were performed in the independent Guangdong case-control population, and only rs2735845 and rs401681 was confirmed to be associated with the risk of NPC (in the combined population from Guangxi and Guangdong:rs2735845, OR=1.23,95% CI=1.12-1.35, P=4.64×10-5 and rs401681, OR= 0.81,95% CI=0.74-0.90, P=1.00 x 10-4). rs2735845 and rs401681 were two independent markers in the association with NPC after corrected by each other. No SNP was significantly associated with the severity of NPC after multiple testing correction. Functional analysis in samples of UC fMSCs showed that compared with CC genotype, the carriers with rs2735845 [G] had a increased level of CLPTM1L mRNA (P =0.02) and rs401681 TT genotype was associated with short telomere length (P=0.002). By immunohistochemistry assay, we found TERT and CLPTM1L were overexpressed in the tissue of NPC, compared with the nasopharyngeal tissue without cancer (both P< 0.001).We found that the short allele carriers (S,272 and 243 bp) of the MNS16A were associated with decreased risk of nasopharyngeal carcinoma (OR=0.86,95% CI= 0.78-0.96, P=0.014) compared with those only have long alleles (L,333 and 302 bp), especially in poorly differentiated squamous cell carcinoma. Subjects with the S allele tended to be older than those with the LL genotypes by age at diagnosis. Furthermore, immunohistochemical analyses showed that the short allele may also have decreased TERT expression in NPC tissues compared with LL genotype (P=0.035).Conclusion:The polymorphisms rs2735845, rs401681 and MNS16A at TERT-CLPTM1L locus on 5p15.33 were confirmed to be associated with the risk of NPC. These findings indicate that the variations at the TERT-CLPTM1L locus on 5p15.33 may confer the susceptibility to NPC.