Abstract
Genital infection with the oncogenic human papillomavirus (HPV) is the necessary cause of cervical cancer and of a large fraction of vulvar cancers. The toll-like receptor (TLR) and the nuclear factor κB (NF-κB) signaling pathways have been implicated in inflammation, autoimmune disease and cancer, but whether common nucleotide variation in these pathways is associated with the risk of cervical and vulvar cancers has received little study. Using data from a population-based case-control study of cervical and vulvar cancers, we genotyped 205 single nucleotide polymorphisms (SNPs) in and around 32 candidate gene regions within these pathways. Gene-based analyses were employed to estimate the associations between individual gene regions and the risk of cervical and vulvar cancers. Odds ratio (OR) and 95% confidence intervals (CI) were calculated to assess the risk of cervical and vulvar cancers for each SNP. P-values were adjusted for multiple testing. A total of 876 cervical cancer cases, 517 vulvar cancer cases and 1,100 controls were included in the analysis. The TNF region was significantly associated with the risks of cervical cancer (gene-based P-value: 2.0×10−4) and vulvar cancer (gene-based P-value: 1.0×10−4). The rare allele (A) of SNP rs2239704 in the 5′ UTR of the LTA gene was significantly associated with increased risks of cervical cancer (OR=1.31, 95% CI: 1.15–1.50; adjusted P-value: 0.013) and vulvar cancer (OR=1.51, 95% CI: 1.30–1.75; adjusted P-value: 1.9×10−5). These findings add to the evidence of the importance of the immune system in the etiology of cervical and vulvar cancers.
Keywords: cervical cancer, vulvar cancer, toll-like receptor, nuclear factor-κB, tumor necrosis factor
Introduction
Genital infection with the oncogenic human papillomavirus (HPV) is the necessary cause of cervical cancer and of a large fraction of vulvar cancers1,2. Although HPV is a common sexually transmitted infection3,4, most women exposed to HPV do not develop cervical or vulvar cancer5, suggesting that other factors must be important for the establishment of a persistent infection that can lead to these cancers. Several inherited genetic factors that influence the host immune response in the presence of HPV have been reported to confer susceptibility to these anogenital cancers6.
Toll-like receptors (TLRs) are transmembrane proteins that recognize specific pathogen-associated molecular patterns (PAMPs) found in viruses and other invading pathogens7,8. Different TLRs are able to identify different PAMPs. Upon activation of the TLRs, a signal transduction is initiated, which involves several adaptor proteins (including TIRAP, TOLLIP, TICAM1 and TICAM2) and several activator proteins (including TANK, IRAK1, IRAK4, TBK1 and IKBKE). This signaling cascade promotes the binding of the nuclear factor-κB (NF-κB) to DNA, which in turn induces cytokine transcription, essential to induce an immune response. An alternative pathway of activation of NF-κB is through the tumor necrosis factor (TNF) superfamily9–11. TNF exercises its function by binding to a member of the TNF receptor superfamily, which includes TNFRSF1A and TNFRSF1B, and the recruitment of several members of the TNF receptor-associated factors (TRAFs) family, such as TANK or TRAF6.
TLRs are thought to be involved in the response to HPV infection12–14. TLR genes are expressed in the female genital tract12,15 and there is some evidence that they may play a role in the clearance of the HPV infection14. HPV oncoproteins E6 and E7 have also been observed to alter the expression of several components of the NF-κB signal pathway in cervical keranocytes16. However, whether the TLR and NF-κB pathways are associated with the risks of cervical and vulvar cancers has not been adequately studied. Therefore, we examined the associations between the common genetic variation in the TLR and NF-κB genetic pathways and cervical and vulvar carcinomas, using data from a population-based case-control study.
Methods
Study Design and Population
The study population and collection of specimens have been described previously17. Briefly, cervical cancer cases were women newly diagnosed with invasive squamous cell carcinoma (SCC) of the cervix (ICD-0 8010–8081) or invasive and in situ adenocarcinoma of the cervix (ICD-0 8140–8560) between January 1986 and June 1998 or between January 2000 and December 2004. They were identified through the Cancer Surveillance System (CSS), a population-based tumor registry that is part of the Surveillance, Epidemiology and End Results program of the National Cancer Institute18. Similarly, vulvar cancer cases were women newly diagnosed with invasive or in situ SCC of the vulva (ICD-0 8010, 8070–8077, 8081) during the same period as the cervical cases and also identified through the CSS. Controls were women without a history of cervical or vulvar cancers, matched to cases on 5-year age groups and county of residency. They were identified by random digit telephone dialing. All women in the study were aged 18–74 years old at the time of diagnosis for the cases or the reference date for the controls. The Institutional Review Board of the Fred Hutchinson Cancer Research Center approved the study.
Data and Specimen collection
In-person interviews were conducted to obtain information on demographic characteristics, reproductive and smoking history and family history of cancers. Blood or buccal samples were collected at the time of the interview.
Genomic DNA was extracted from buffy coats from blood samples or from cell pellets from buccal samples. Archival tissue blocks from biopsy or surgery were retrieved to determine the presence and type of HPV DNA in the tumors of cervical and vulvar cases using a polymerase chain reaction (PCR)19.
SNP Selection and Genotyping
Thirty-two candidate genes related to the TLR and NF-κB genetic pathways were included in this study (AZI2, IKBKE, IRAK1, IRAK4, IRF3, LST1, LTA, LTB, MAP3K1, MAP3K7, NCR3, NFKB1, NFKB2, RELA, RELB, TANK, TBK1, TICAM1, TICAM2, TIRAP, TLR3, TLR4, TLR7, TLR9, TNF, TNFRSF1A, TNFRSF1B, TOLLIP, TRAF3, TRAF6, VISA and ZBP1). Tagging single nucleotide polymorphisms (tagSNPs) covering the exons, introns and the 3′ and 5′ 4kb flanking sequences were selected for each of the above genes. Because of the proximity of genes LST1, LTA, LTB, NCR3 and TNF and the fact that the flanking sequences overlap, they were collectively named the TNF-region. Using information from the CEU-HapMap population, the SeattleSNPs Variation Discovery Resource (http://gvs.gs.washington.edu) and the Snagger algorithm20, tagSNPs were selected based on having a pairwise r2≥0.80 and minor allele frequency (MAF) greater than 5% for each gene.
A total of 261 tagSNPs were genotyped using the Illumina Goldengate multiplex platform (Illumina Inc., San Diego, USA) or the KASPar platform (KBioscience, Hoddesdon, UK). Forty-two tagSNPs failed genotyping and 5 were monomorphic. Of the remaining 214 tagSNPs, 9 tagSNPs had MAF less than 1% in our control population and were excluded from the analysis, leaving 205 tagSNPs in 28 candidate gene regions (Supplemental Table 1).
Data Analysis
Analyses were restricted to self-reported Caucasian subjects (88.8% of cervical cases, 95.1% of vulvar cases and 91.8% of controls). We also excluded women with zero lifetime sex partners (0.2% of cervical cases, 0.2% of vulvar cases and 1.3% of controls) so that women could have been exposed to oncogenic HPV. Hardy-Weinberg equilibrium was calculated among controls using an exact test21 (Supplemental Table 1). After correcting for multiple testing, none of the tagSNP genotypes showed evidence of deviation from Hardy-Weinberg equilibrium. Linkage disequilibrium (LD) was estimated in our control population using the r2 measure among the 205 tagSNPs (Supplemental Figure 1). Associations between gene regions and disease outcome were computed by combining SNP-based p-values by means of an adaptive rank truncated product22. This method takes into account the LD structure of the SNPs within a gene region to compute the gene-based p-value. Significance of the gene-based P-value was assessed using a permutation procedure with 10,000 permutations. SNP-based p-values, odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between SNPs and cancer were computed using unconditional logistic regression assuming an additive model (coded as 0, 1, 2) and adjusted for age as a continuous variable. Due to X inactivation, analyses of tagSNPs in the X chromosome were restricted to women with homozygous genotypes. Adjusted p-values were gene-based p-values or SNP-based p-values adjusted for multiple testing using the Bonferroni method. Analyses were carried out using the R software environment (version 2.15.0 for Macintosh).
Results
A total of 876 cervical cases, 517 vulvar cases and 1,100 controls were included in the analysis. Of the 876 cervical cases, 403 (46%) were diagnosed with SCC and 473 (54%) with adenocarcinoma. Of the 517 women with vulvar cancer, 429 (83%) were tested for HPV DNA, with the majority of them testing positive (350, 82%) and only a small fraction testing negative (79, 18%). Cervical cases were younger, current smokers and had more lifetime sex partners compared to controls (Supplemental Table 2). Vulvar cases were slightly older, more likely to be current smokers, had less education, and a higher number of lifetime sex partners than controls.
Gene-based analysis identified the TNF region to be significantly associated with the risks of cervical cancer (gene-based P-value: 2.0×10−4; Table 1) and vulvar cancer (gene-based P-value: 1.0×10−4). The association remained significant after Bonferroni adjustment for multiple testing and further adjustment for smoking and lifetime number of sex partners (results not shown). When analyses were stratified by histological types, the TNF region was still significantly associated with the risk of cervical SCC (gene-based P-value: 1.5×10−3) and with HPV positive vulvar cancer (gene-based P-value: 1.0×10−4). Other gene regions did not reach statistical significance after correcting for multiple testing.
Table 1.
Gene-based association analysis for cervical and vulvar cancers, Seattle-Puget Sound Region, 1986–2004.
| Gene region | Chr. | Number of SNPs within gene region | Cervical cancer
|
Vulvar cancer
|
||||
|---|---|---|---|---|---|---|---|---|
| All cases P-value | SCC P-value | Adenocarcinoma P-value | All cases P-value | HPV+ P-value | HPV- P-value | |||
| TNFRSF1B | 1 | 18 | 0.967 | 0.797 | 0.956 | 0.144 | 0.285 | 0.018 |
| IKBKE | 1 | 19 | 0.021 | 0.143 | 0.038 | 0.983 | 0.950 | 0.314 |
| TRAF2 | 2 | 6 | 0.281 | 0.531 | 0.220 | 0.077 | 0.047 | 0.697 |
| AZI2 | 3 | 4 | 0.315 | 0.531 | 0.503 | 0.664 | 0.885 | 0.586 |
| TLR9 | 3 | 2 | 0.223 | 0.306 | 0.101 | 0.116 | 0.218 | 0.031 |
| NFKB1 | 4 | 14 | 0.912 | 0.925 | 0.559 | 0.469 | 0.643 | 0.241 |
| TLR3 | 4 | 11 | 0.412 | 0.660 | 0.524 | 0.512 | 0.746 | 0.777 |
| MAP3K1 | 5 | 1 | 0.148 | 0.532 | 0.112 | 0.823 | 0.850 | 0.756 |
| TICAM2 | 5 | 7 | 0.906 | 0.689 | 0.963 | 0.441 | 0.695 | 0.224 |
| TNF-region | 6 | 12 | 2.0×10−4 | 1.5×10−3 | 0.013 | 1.0×10−4 | 1.0×10−4 | 0.506 |
| MAP3K7 | 6 | 6 | 0.363 | 0.548 | 0.034 | 0.986 | 0.723 | 0.908 |
| TLR4 | 9 | 12 | 0.905 | 0.859 | 0.712 | 0.418 | 0.546 | 0.653 |
| NFKB2 | 10 | 3 | 0.005 | 0.034 | 0.045 | 0.340 | 0.817 | 0.106 |
| TOLLIP | 11 | 17 | 0.337 | 0.229 | 0.609 | 0.515 | 0.909 | 0.211 |
| TRAF6 | 11 | 4 | 0.379 | 0.079 | 0.712 | 0.952 | 0.919 | 0.439 |
| RELA | 11 | 3 | 0.358 | 0.257 | 0.590 | 0.011 | 0.003 | 0.590 |
| TIRAP | 11 | 8 | 0.199 | 0.743 | 0.238 | 2.6×10−3 | 0.040 | 0.378 |
| TNFRSF1A | 12 | 5 | 0.655 | 0.170 | 0.982 | 0.550 | 0.507 | 0.052 |
| IRAK4 | 12 | 4 | 0.981 | 0.993 | 0.919 | 0.262 | 0.290 | 0.771 |
| TBK1 | 12 | 5 | 0.238 | 0.526 | 0.216 | 0.994 | 0.641 | 0.198 |
| TRAF3 | 14 | 12 | 0.717 | 0.915 | 0.346 | 0.245 | 0.246 | 0.923 |
| TICAM1 | 19 | 4 | 0.759 | 0.484 | 0.189 | 0.887 | 0.787 | 0.530 |
| RELB | 19 | 4 | 0.860 | 0.547 | 0.300 | 0.655 | 0.413 | 0.116 |
| IRF3 | 19 | 3 | 0.833 | 0.284 | 0.303 | 0.109 | 0.402 | 0.096 |
| VISA | 20 | 7 | 0.823 | 0.544 | 0.739 | 0.852 | 0.641 | 0.917 |
| ZBP1 | 20 | 7 | 0.184 | 0.490 | 0.175 | 0.549 | 0.083 | 0.506 |
| TLR7 | 23 | 7 | 0.561 | 0.538 | 0.241 | 0.007 | 0.004 | 0.113 |
Number of controls: 1,100; number of cervical cases: 886; number of vulvar cases: 517. Bold indicates significant at the 5% level. P-values less than 0.05/27×1.9κ10−3 were statistically significant after Bonferroni adjustment for multiple testing.
Of the twelve tagSNPs in the TNF region, several were associated with the risks of cervical and vulvar cancers. The rare allele (A) of the LTA (lymphotoxin alpha) rs2239704 5′ untranslated region (UTR) tagSNP was significantly associated with a 31% increased risk of cervical cancer (OR=1.31, 95% CI: 1.15–1.50; adjusted P-value: 0.013; Table 2) and with a 51% increased risk of vulvar cancer (OR=1.51, 95% CI: 1.30–1.75; adjusted P-value: 1.9×10−5; Table 3). When cervical cancer was stratified by histological type, there was still a 31% increased risk for SCC (OR=1.31, 95% CI: 1.11–1.55; adjusted P-value: 0.342) and adenocarcinoma (OR=1.31, 95% CI: 1.11–1.54; adjusted P-value: 0.298). When vulvar cancer was stratified by HPV status, the A allele of SNP rs2239704 was associated with a significant 58% increased risk of HPV-positive vulvar cancer (OR=1.58, 95% CI: 1.33–1.87; adjusted P-value: 5.1×10−5), while it was associated with a non-significant 24% increased risk of HPV-negative vulvar cancer (OR=1.24, 95% CI: 0.89–1.73; adjusted P-value: 1.00).
Table 2.
Association between tagSNPs in the TNF region and cervical cancer risk, Seattle-Puget Sound Region, 1986–2004.
| SNPs | MAF Controls (N=1,100) (%) | All cervical cancer
|
Cervical SCC
|
Cervical adenocarinoma
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MAF Cases (N=876) (%) | OR* | (95% CI) * | Adj. P‡ | MAF Cases (N=403) (%) | OR* | (95% CI) * | Adj. P‡ | MAF Cases (N=473) (%) | OR* | (95% CI) * | Adj. P‡ | ||
| rs2009658 | 17.12 | 13.00 | 0.72 | (0.59–0.86) | 0.091 | 11.75 | 0.64 | (0.5–0.82) | 0.101 | 14.03 | 0.79 | (0.63–0.99) | 1.000 |
| rs915654 | 36.25 | 30.68 | 0.79 | (0.69–0.91) | 0.157 | 31.41 | 0.81 | (0.68–0.97) | 1.000 | 30.09 | 0.77 | (0.65–0.92) | 0.608 |
| rs2239704 | 37.65 | 43.80 | 1.31 | (1.15–1.50) | 0.013 | 43.98 | 1.31 | (1.11–1.55) | 0.342 | 43.66 | 1.31 | (1.11–1.54) | 0.298 |
| rs2229094 | 27.92 | 22.38 | 0.72 | (0.62–0.84) | 0.005 | 20.94 | 0.67 | (0.55–0.82) | 0.020 | 23.55 | 0.77 | (0.64–0.93) | 1.000 |
| rs2229092 | 6.95 | 5.89 | 0.83 | (0.63–1.09) | 1.000 | 5.11 | 0.71 | (0.49–1.03) | 1.000 | 6.53 | 0.95 | (0.69–1.32) | 1.000 |
| rs1799964 | 23.08 | 18.18 | 0.72 | (0.61–0.85) | 0.016 | 16.71 | 0.66 | (0.53–0.82) | 0.036 | 19.38 | 0.77 | (0.63–0.94) | 1.000 |
| rs1800610 | 7.53 | 9.29 | 1.24 | (0.99–1.55) | 1.000 | 9.53 | 1.27 | (0.96–1.69) | 1.000 | 9.10 | 1.22 | (0.93–1.6) | 1.000 |
| rs3093662 | 7.95 | 6.41 | 0.74 | (0.57–0.96) | 1.000 | 5.87 | 0.69 | (0.49–0.98) | 1.000 | 6.85 | 0.78 | (0.57–1.07) | 1.000 |
| rs769178 | 7.26 | 9.19 | 1.27 | (1.01–1.60) | 1.000 | 9.37 | 1.29 | (0.97–1.73) | 1.000 | 9.05 | 1.26 | (0.96–1.66) | 1.000 |
| rs2256965 | 40.26 | 44.59 | 1.20 | (1.06–1.37) | 1.000 | 45.17 | 1.22 | (1.04–1.44) | 1.000 | 44.11 | 1.19 | (1.01–1.39) | 1.000 |
| rs1052248 | 29.45 | 25.38 | 0.80 | (0.69–0.92) | 0.476 | 23.82 | 0.74 | (0.61–0.9) | 0.445 | 26.66 | 0.84 | (0.71–1.01) | 1.000 |
| rs11575839 | 2.47 | 1.66 | 0.67 | (0.41–1.09) | 1.000 | 1.66 | 0.66 | (0.35–1.26) | 1.000 | 1.67 | 0.69 | (0.38–1.25) | 1.000 |
MAF: Minor allele frequency.
Age-adjusted OR assuming an additive model.
P value adjusted for multiple testing using the Bonferroni adjustment across all 205 SNPs. Bold indicates significant at the adjusted 5% level.
Table 3.
Association between tagSNPs in the TNF region and vulvar cancer risk, Seattle-Puget Sound Region, 1986–2004.
| SNPs | MAF Controls (N=1,100) (%) | All vulvar cancer
|
Vulvar cancer (HPV +)
|
Vulvar cancer (HPV -)
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MAF Cases (N=517) (%) | OR* | (95% CI) * | Adj. P‡ | MAF Cases (N=403) (%) | OR* | (95% CI) * | Adj. P‡ | MAF Cases (N=473) (%) | OR* | (95% CI) * | Adj. P‡ | ||
| rs2009658 | 17.12 | 13.09 | 0.72 | (0.58–0.90) | 0.652 | 12.79 | 0.70 | (0.55–0.90) | 1.000 | 16.67 | 0.93 | (0.60–1.45) | 1.000 |
| rs915654 | 36.25 | 28.38 | 0.70 | (0.59–0.82) | 3.2×10−3 | 27.09 | 0.65 | (0.54–0.79) | 2.2×10−3 | 33.33 | 0.87 | (0.61–1.22) | 1.000 |
| rs2239704 | 37.65 | 47.95 | 1.51 | (1.30–1.75) | 1.9×10−5 | 49.00 | 1.58 | (1.33–1.87) | 5.1×10−5 | 42.95 | 1.24 | (0.89–1.73) | 1.000 |
| rs2229094 | 27.92 | 22.32 | 0.74 | (0.62–0.88) | 0.172 | 21.63 | 0.71 | (0.58–0.87) | 0.216 | 26.92 | 0.96 | (0.66–1.39) | 1.000 |
| rs2229092 | 6.95 | 5.31 | 0.75 | (0.54–1.03) | 1.000 | 5.86 | 0.83 | (0.58–1.19) | 1.000 | 5.77 | 0.81 | (0.41–1.62) | 1.000 |
| rs1799964 | 23.08 | 18.71 | 0.76 | (0.63–0.92) | 1.000 | 18.77 | 0.77 | (0.62–0.96) | 1.000 | 20.51 | 0.86 | (0.57–1.29) | 1.000 |
| rs1800610 | 7.53 | 10.53 | 1.44 | (1.12–1.86) | 0.974 | 10.17 | 1.38 | (1.04–1.85) | 1.000 | 8.97 | 1.22 | (0.70–2.13) | 1.000 |
| rs3093662 | 7.95 | 7.02 | 0.88 | (0.66–1.18) | 1.000 | 7.16 | 0.90 | (0.64–1.26) | 1.000 | 5.77 | 0.76 | (0.38–1.54) | 1.000 |
| rs769178 | 7.26 | 10.72 | 1.53 | (1.18–1.97) | 0.233 | 10.46 | 1.48 | (1.11–1.97) | 1.000 | 8.97 | 1.27 | (0.73–2.20) | 1.000 |
| rs2256965 | 40.26 | 49.41 | 1.43 | (1.23–1.66) | 5.5×10−4 | 50.29 | 1.48 | (1.25–1.76) | 1.4×10−3 | 47.44 | 1.32 | (0.95–1.83) | 1.000 |
| rs1052248 | 29.45 | 24.07 | 0.76 | (0.64–0.91) | 0.414 | 23.35 | 0.73 | (0.60–0.89) | 0.465 | 27.56 | 0.92 | (0.64–1.33) | 1.000 |
| rs11575839 | 2.47 | 2.33 | 0.95 | (0.57–1.57) | 1.000 | 2.69 | 1.09 | (0.63–1.90) | 1.000 | 0.65 | 0.25 | (0.03–1.86) | 1.000 |
MAF: Minor allele frequency.
Age-adjusted OR assuming an additive model.
P value adjusted for multiple testing using the Bonferroni adjustment across all 205 SNPs. Bold indicates significant at the adjusted 5% level.
The rare allele (G) of LTA the rs2229094 missense tagSNP was significantly associated with a decreased risk of cervical cancer (OR=0.72, 95% CI: 0.62–0.84; adjusted P-value: 0.005). This significant association was present for cervical SCC (OR=0.72, 95% CI: 0.62–0.84; adjusted P-value: 0.005). This allele was also associated with a decreased risk of cervical adenocarcinoma (OR=0.77, 95% CI: 0.64–0.93; adjusted P-value: 1.000), vulvar cancer (OR=0.74, 95% CI: 0.62–0.88; adjusted P-value: 0.172), and HPV positive vulvar cancer (OR=0.71, 95% CI: 0.58–0.87; adjusted P-value: 0.216), albeit not significant after correcting for multiple testing. Similar associations were found for the rare allele (G) of the TNF rs1799964 flanking 5′ UTR tagSNP. The pairwise LD between tagSNPs rs2229094 and rs1799964 was r2=0.76.
The minor allele (A) of LTA rs915654 flanking 5′ UTR tagSNP was significantly associated with a decreased risk of vulvar cancer (OR=0.70, 95% CI: 0.59–0.72; adjusted P-value: 3.2×10−3) and of HPV positive vulvar cancer (OR=0.65, 95% CI: 0.54–0.79; adjusted P-value: 2.2×10−3). The strength of the association was weaker for HPV negative vulvar cancer and cervical cancer (Tables 2 and 3).
Finally, the minor allele (A) of the LST1 (leukocyte specific transcript 1) rs2256965 intronic tagSNP was significantly associated with an increased risk of vulvar cancer (OR=1.43, 95% CI: 1.23–1.66; adjusted P-value: 5.5×10−4) and of HPV positive vulvar cancer (OR=1.48, 95% CI: 1.25–1.76; adjusted P-value: 1.4×10−3). The increased risk was also present for HPV negative vulvar cancer and cervical cancer (overall and for SCC and adenocarcinoma), but they did not reach statistical significance after Bonferroni correction (Tables 2 and 3).
Associations between other SNPs and the risks of cervical and vulvar cancers are presented in Supplemental Tables 3–8.
Discussion
In this study of the TLR and NF-κB genetic pathways on the risk of cervical and vulvar cancers, we found some evidence that common variation in the TNF region was significantly associated with the risks of both types of malignancies. In particular, within the TNF-region, we found that the SNP rs2239704, located in the 5′ UTR of the LTA gene, was significantly associated with increased risks of cervical and vulvar cancers.
The TNF gene region is a rich gene locus that includes LTA upstream of TNF and LTB (lymphotoxin beta), LST1 and NCR3 (natural cytotoxicity triggering receptor 3) downstream of TNF. LTA and LTB belong to the TNF superfamily (members 1 and 3, respectively) and reside in the major histoccompatiblity complex (MHC) on chromosome 6, variation in which has also been found to be associated with cervical cancer23–25. To better understand the genetic relationship between the TNF-region and other areas within the MHC region, we computed the LD structure between tagSNPs in five major loci within the MHC (HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1) and the SNPs in the TNF-region presented in our study using data from 40 women with European ancestry downloaded from the 1000 genomes (http://www.1000genomes.org) (Supplemental Figure 2). There was little evidence of high linkage between the tagSNPs in the MHC and the tagSNPs in the TNF-region analyzed (all pairs between the two regions had r2<0.379). However, this was a small dataset and information on some MHC tagSNPs was not available in the 1000 genomes dataset. In addition, most HLA alleles are not well captured using tagSNPs, and a more focused studied is needed.
LTA codes for a cytokine produced by lymphocytes and plays an important role in the genesis of lymphoid organs26. Genetic variation at the LTA/TNF/LTB locus has been associated with several infectious diseases, such as the denge virus27, malaria 28, influenza 29 or sepsis 30 as well as with non-Hodgkin’s lymphoma 31,32.
In contrast to our findings, a previous study reported that 5′ UTR SNP rs2239704 was not associated with HPV persistence or progression to cervical cancer in a Costa Rican population33. This could be due to the lower power in the previous study (469 women with cancer, 390 with persistent HPV infection and 452 controls), to the ethnically distinct study populations, or to the potential involvement of this SNP in the initial response to infection, rather than at later stages of carcinogenesis.
The G allele of rs2229094, a missense mutation Cys13Arg in the LTA gene, was significantly associated with a 30% decreased risk of cervical cancer in our study. Although the association was not found to be significant with vulvar cancer, it was of the same magnitude and direction. This SNP was found to be associated with cancer-specific mortality in a recent prospective study 34. In addition, SNP rs1799964, which is in moderate to high LD with rs2229094 (r2=0.76 in our control population), has also been inversely related to smoking related cancers35, and cigarette smoking is a risk factor for SCC cervical and vulvar cancers.
Finally, the G allele of rs2256965, a SNP located in an intronic region of the LST1 gene, was significantly associated with a 40% increased risk of vulvar cancer, but to a lesser extent with cervical cancer. The protein encoded by the LST1 gene can inhibit the proliferation of lymphocytes, potentially preventing a competent immune response to the HPV virus. This gene, in combination with HLA genes, has been found to be important in the immune response to rubella vaccine 36.
The role of genetic variation in the TLR genes on the risk of cervical or vulvar cancer has not been well-studied. We did not observe any significant association between tagSNPs in TLR coding genes and the risk of these cancers. A prior study looked at two SNPs on TLR4 in relation to persistence or progression of cervical cancer but did not report assocations33. However, the same study also found IRF3 SNP rs7251 to be associated with persistence, which were not associated with cervical and vulvar risk in our study.
The main limitation of our study was the relatively small number of cases and controls which could have prevented us from detecting weaker associations between SNPs and cervical or vulvar cancer. Due to the large number of SNPs that we studied, some of the reported associations could have been due to chance. However, we corrected for multiple testing and focused our discussion on the gene region that was associated with both HPV-related cancers. Further, the individual SNPs reported here had similar associations, in direction and magnitude, for cervical and vulvar cancers. A strength of our study was the use of gene-set analysis that allowed us to maximize our ability to detect effects that are only significant after combining the small effects of individual SNPs.
In summary, our study underscores the role of genetic variation of immune genes in the development of HPV related cervical and vulvar cancers. Future studies should further examine the role of the TNF region in the cervical and vulvar cancer etiology.
Supplementary Material
Research novelty.
The toll-like receptor and the nuclear factor κB signaling pathways are thought to be involved in response to the human papillomavirus infection. Here, the authors investigate whether genetic variation in these pathways may confer susceptibility to cervical and vulvar cancers. They found that the TNF-region was significantly associated with both cancers. A single nucleotide polymorphism in this region was found to significantly increase cervical cancer risk by 31% and vulvar cancer risk by 51%.
Acknowledgments
This work was supported by the research grants (R01CA112512, P01CA042792), contracts (NO1-CN-67009, NO1-PC-35142) and a training grant (T32CA009168 to CB) from the National Cancer Institute, as well as institutional funding from the Fred Hutchinson Cancer Research Center.
Footnotes
Conflict of interest: The authors declare that they have no conflict of interest.
References
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