A Molecular Epidemiology Study On The Association Of IL12A, IL12B And TLR9 Genes Polymorphisms And The Susceptibility Of Cervical Cancer

Cervical cancer is the second most common cancer among women worldwide, with an estimated 493,000 new cases and 274,000 deaths in the year 2002 and 83% of the cases occurred in developing countries. Although there have been quite substantial declines both in incidence and mortality rates in the past 30 years, more than 100,000 cervical cancer cases take place in China every year. Epidemiological and laboratory-based studies have identified the infection with one of 15 high-risk, or oncogenic, HPV types as a necessary but not sufficient cause of cervical cancer. The prevalence of genital HPV infections in developing countries is very high in young women, but most of the infections regress without intervention. Given the mounting evidence that long-term HPV infection is a prerequisite for cervical carcinogenesis, host genetic variations that influence the primary immune response may determine the outcome of high risk HPV infection. Thus, it is crucial to investigate genetic risk factors which closely related with immune response to HPV clearance in cervical cancer development in general population.Mounting evidence indicates Th type 1 (Th1) cells, which promote cell-mediated immunity (CMI) responses to intracellular pathogens by cytokine production, are necessary for the clearance of HPV. Interleukin-12 (IL12) is a heterodimeric pro-inflammatory cytokine formed by a 35-kDa light chain (known as p35 encoded by IL12A) and a 40-kDa heavy chain (known as p40 encoded by IL12B), which induces the production of interferon-gamma (IFN-γ), favors the differentiation of Th1 cells and forms a link between innate resistance and adaptive immunity. Studies showed that IL12 had a central role in Th1 responses. Besides the activity of antivirus, IL12 is also important for host resistance to tumors. The antitumor activity of IL12 has been extensively reported in mouse models of cancer, where it has been shown to inhibit tumorigenesis and induce regression of established tumors. The major antitumor activities of IL12 rely on its ability to promote Th1 adaptive immunity and CTL responses. Studies showed the polymorphisms in the IL12A and IL12B gene are associated with risk of cancer. TLRs play a key role in host defense during pathogen infection by regulating and linking the innate and Th1 immune responses. They are mainly expressed on antigen-presenting cells (APCs) of the innate immune system and play important roles in recognizing specific foreign pathogen components. TLR9 is able to recognize dsDNA-derived CpG motifs and induce inflammatory cytokines such as IL12 to promote Th1 adaptive immunity, which is the known mechanism to HPVs immunity. Thus, we conducted a case-control study aiming to investigate the association of SNPs of IL12A, IL12B and TLR9 genes with cervical cancer risk in Chinese women. Part I IL12A and IL12B Potentially Functional SNPs and Risk of Cervical Cancer in Chinese WomenThe two subunits of IL12, p35 and p40 encoded by IL12A and IL12B, respectively, are located on separate chromosomes (3p12–q13.2 and 5q31–33). Because of the functional relevance of IL12, several molecular epidemiological studies were performed to investigate the relationship between the IL12A and IL12B polymorphisms and risk of cancers, including non-Hodgkin lymphoma, hepatocellular carcinoma, lung cancer, gastric cancer and others. Few studies evaluated IL12A and IL12B polymorphisms and risk of cervical cancer. In the present study, we searched all the common potentially functional SNPs in IL12A and IL12B. As a result, we selected three common SNPs, that is, rs2243115 (5'UTR T>G) and rs568408 (3'UTR G>A) in IL12A and rs3212227 (3'UTR A>C) in IL12B. The aim of this case-control study is to investigate the associations of the three SNPs with cervical cancer risk. This case-control study included 404 histologically confirmed incident cervical cancer patients and 404 cancer-free controls frequency-matched for age and area. We genotyped the three polymorphisms by PCR-RFLP (PCR- Restriction Fragment Length Polymorphism) and PCR-PIRA (PCR-PrimerIntroduced Restriction Analysis) method. In addition,genotyping was performed blindly and 10% of the samples were randomly selected for repeated assays. In this case-control study, we found that the IL12A rs568408 GA/AA and IL12B rs3212227 AC/CC variant genotypes were associated with a significantly increased risk of cervical cancer [adjusted odds ratio (OR) = 1.43, 95% confidence interval (CI) = 1.06-1.93 and adjusted OR = 1.30, 95% CI = 0.97-1.75, respectively], compared with their corresponding wild-type homozygotes. Moreover, a significant gene-gene interaction of these two loci were evident in the risk of cervical cancer; subjects carrying variant genotypes of both loci had a 1.82-fold (95% CI = 1.28-2.57) increased risk of cervical cancer. In the stratified analyses, the combined genetic effect was more pronounced in patients with early-stage tumors and those with more parities. Subjects carrying rs568408 AG/AA and rs3212227 AC/CC genotypes and having more than 2 parities showed a 6.00-fold (95% CI = 2.86-12.56) elevated cervical cancer risk (P for multiplicative interaction = 0.046). These findings indicated that IL12A rs568408 and IL12B rs3212227 may individually and jointly contribute to the risk of cervical cancer, and may modify cervical cancer risk associated with parity, but these data need further validation.Part II TLR9 Promoter SNP and Risk of Cervical Cancer in Chinese WomenStudies showed TLR9 is able to recognize DNA CpG motifs from HPV16, and promote the Th1 adaptive immunity by inducing cytokines, such as IL12. However, E6 and E7 oncoproteins can down-regulate the activity of the TLR9 promoter and deregulate the TLR9 transcript in vivo and in vitro, which may be one of the major mechanism for HPV to escape the immune surveillance and to be persistant infection. So, we hypothesized that the polymorphisms of TLR9, especially which in promoter may be associated with cervical cancer risk in Chinese women. Useing the potentially fuctional SNPs selection stratege, we selected the SNP -1486T/C (rs187084) in promoter region. This case-control study included 633 histologically confirmed incident cervical cancer patients and 633 cancer-free controls frequency-matched for age and area. We genotyped the polymorphism by PCR-RFLP method. Genotyping was performed blindly and 10% of the samples were randomly selected for repeated assays.In this case-control study, we found that -1486CT genotype was associated with a significantly increased risk of cervical cancer [adjusted odds ratio (OR) = 1.29, 95% confidence interval (CI) = 1.00-1.64] and the -1486CT/CC variant genotypes were associated with a non-significantly increased risk of cervical cancer [adjusted odds ratio (OR) = 1.24, 95% confidence interval (CI) = 0.98-1.56], compared with the wild-type homozygote -1486TT. In the stratified analyses, the -1486CT/CC variant genotypes effect was more pronounced in patients with early-stage tumors and those with more paritiesThese results supported that TLR9 -1486T/C (rs187084) might be associated with cervical cancer development in Chinese population.