【作者】 张旸；

【导师】 贺福初； 周钢桥；

【作者基本信息】 北京协和医学院 ， 遗传学， 2011， 博士

【摘要】 目的：TERT-CLPTM1L基因区域多态性已被报道与多种肿瘤的发生风险相关。该区域存在两个已知的基因,端粒酶反转录酶(telomerase reverse transcriptase, TERT)和唇腭裂跨膜1样蛋白(cleft lip and palate transmembrane 1 like, CLPTM1L)基因。鉴于这两个基因的生化、生理功能和肿瘤病理学意义,这一基因区域是生物学证据非常充分的鼻咽癌易感基因候选位点,因此本研究探讨了这一基因区域多态性与鼻咽癌遗传易感性的关系。方法：采用生物信息学与文献调研相结合的方法,根据中国汉族人群(CHB人群)HapMap II数据,在TERT-CLPTM1L基因区域选择单倍型标签单核苷酸多态性(htSNPs)位点。采用中通量的基于Sequenom质谱技术和SNPstream的分型方法,以及TagMan法和聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)分型方法,在广西鼻咽癌病例对照人群(病例855人,对照1036人)中对htSNPs进行了分型,以多因素logistic回归方法分析htSNPs与鼻咽癌的发生风险和病程进展的相关性。在独立人群——广东鼻咽癌病例对照人群(病例997人,对照972人)及广西鼻咽癌核心家系人群中对上述阳性关联的SNP位点进行验证。用生物信息学方法对在多个人群中均呈阳性关联的位点进行功能预测,用双荧光素酶报告基因实验研究阳性关联的位点不同基因型之间是否具有增强子活性的差异,并研究不同等位型之间在端粒长度、TERT mRNA及蛋白表达水平.CLPTM1L mRNA及蛋白表达水平的差异。此外,还研究了TERT下游的功能性串联重复多态性MNS16A与鼻咽癌发生发展的相关性。采用PCR联合电泳的方法,在广西鼻咽癌病例对照人群中对TERTMNS16A进行分型。以多因素logistic回归方法分析MNS16A与鼻咽癌的发生风险和病程进展的相关性。结果：在TERT-CLPTM1L基因区域共确定了26个htSNPs。在广西鼻咽癌病例对照人群中分型成功25个位点,统计分析去除分布不符合哈迪-温伯格平衡准则的位点和最小等位频率(MAF)小于0.01的位点3个。以多因素logistic回归方法分析,在校正年龄、性别、吸烟、吸烟量、饮酒、家族史后,经过SNPSpD多重检验校正,在加性遗传模式和共显性遗传模式下共有4个SNPs (rs2736098、rs2735845、rs402710和rs401681)与鼻咽癌的发生风险相关。对这4个SNPs在广东鼻咽癌病例对照人群中进行了验证,发现rs2735845和rs401681仍然与鼻咽癌的发生风险显著相关(广西广东联合人群中rs2735845, OR=1.23,95% CI=1.12-1.35, P= 4.64×10-5; rs401681, OR=0.81,95% CI=0.74-0.90, P=1.00×10-4)。而且这两个位点相互独立,与环境因素不存在交互作用。22个htSNPs中没有发现与鼻咽癌病程进展相关的位点。功能实验显示,在脐带来源的间充质干细胞中,与CC基因型相比,rs2735845 G等位携带者的CLPTM1L mRNA水平显著增加(P=0.02)；与CC基因型相比,rs401681 TT基因型与短的端粒长度显著相关(P=0.002)。免疫组化实验显示,TERT、CLPTM1L在鼻咽癌组织中表达均高于非癌鼻咽部组织。对TERT功能性串联重复MNS16A进行分型,经多因素logistic回归分析发现MNS16A S等位型(272和243bp)是鼻咽癌发生风险的保护等位型(OR=0.86,95%CI=0.78-0.96,P=0.014),而且在分化差的鳞状细胞癌组中其保护作用更显著。此外,携带S等位的鼻咽癌患者年龄较LL基因型(333/302和302/302bp)鼻咽癌患者年龄大。S等位为鼻咽癌独立的保护因素,与年龄、性别、吸烟、吸烟量、饮酒及家族史无交互作用。MNS16A与鼻咽癌严重程度不相关。通过免疫组化实验,比较MNS16A基因型之间TERT的蛋白表达水平,发现携带S等位型的个体其TERT的表达显著低于携带LL基因型的个体(P=0.035)。结论：TERT-CLPTM1L基因区域的多态性位点与鼻咽癌的易感性显著相关。本研究首次鉴定出了与鼻咽癌遗传易感性关联的TERT-CLPTM1L基因区域中的两个单核苷酸多态性位点(rs2735845和rs401681)和一个串联重复多态性位点(MSN16A)。本研究为鼻咽癌的遗传易感性提供了新的遗传标记,有助于鼻咽癌发病机理的进一步研究和鼻咽癌的早期预防、诊断和个体化治疗。更多还原

【Abstract】 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.更多还原