Conceived and designed the experiments: SW PG KY CP MS ACR MS CB MS SW RDB RH SJC AH. Performed the experiments: SW RDB SJC. Analyzed the data: SW KY QL AH. Contributed reagents/materials/analysis tools: KY QL SW. Wrote the paper: SW KY MS MS AH.
The authors have declared that no competing interests exist.
HPV infrequently persists and progresses to cervical cancer. We examined host genetic factors hypothesized to play a role in determining which subset of individuals infected with oncogenic human papillomavirus (HPV) have persistent infection and further develop cervical pre-cancer/cancer compared to the majority of infected individuals who will clear infection.
We evaluated 7140 tag single nucleotide polymorphisms (SNPs) from 305 candidate genes hypothesized to be involved in DNA repair, viral infection and cell entry in 416 cervical intraepithelial neoplasia 3 (CIN3)/cancer cases, 356 HPV persistent women (median: 25 months), and 425 random controls (RC) from the 10,049 women Guanacaste Costa Rica Natural History study. We used logistic regression to compute odds ratios and p-trend for CIN3/cancer and HPV persistence in relation to SNP genotypes and haplotypes (adjusted for age). We obtained pathway and gene-level summary of associations by computing the adaptive combination of p-values. Genes/regions statistically significantly associated with CIN3/cancer included the viral infection and cell entry genes 2′,5′ oligoadenylate synthetase gene 3 (
Persistent infection with one of approximately 15 types of human papillomavirus (HPV) is necessary for the development of cervical cancer and its immediate precursor, cervical intraepithelial neoplasia grade 3 (CIN3). However, HPV infection is not a sufficient cause of cervical cancer/CIN3 and HPV cofactors have been identified, including oral contraceptive use and smoking
We evaluated data on 7,140 candidate single nucleotide polymorphisms (SNP) in 305 candidate genes/regions (161 viral infection and cell entry and 144 DNA repair genes). All known DNA repair genes were included
We found the DNA repair pathway as a whole statistically significantly associated with CIN3/cancer (p = 0.0197) and with HPV persistence (p = 0.0472) but not progression (p = 0.7451) (
DNA Repair Pathway | Genes | # genes | # SNPs | (i) CIN3/CA vs RC | (ii) CIN3/CA vs HPV persisters | (iii) HPV persisters vs RC |
All genes | 138 | 2985 | 0.7451 | |||
Base excision repair | 17 | 363 | 0.1961 | 0.6703 | 0.1609 | |
Chromatin Structure | 2 | 27 | 0.6365 | 0.0912 | 0.3588 | |
DNA polymerases | 14 | 289 | 0.2588 | 0.7481 | 0.6309 | |
Direct reversal of damage | 2 | 105 | 0.9515 | 0.9996 | 0.9975 | |
Editing/processing nucleases | 6 | 95 | 0.1961 | 0.3546 | ||
Genes defective in diseases associated with sensitivity to DNA damaging agents | 4 | 101 | 0.2465 | 0.2051 | 0.0732 | |
Fanconi anemia | 10 | 222 | 0.6062 | 0.2230 | ||
Homologous recombination | 17 | 364 | 0.1185 | 0.7500 | 0.2318 | |
Mismatch excision repair | 10 | 254 | 0.7061 | 0.1263 | 0.4253 | |
Modulation of nucleotide pools | 3 | 51 | 0.6242 | 0.2219 | ||
Non-homologous end-joining | 5 | 200 | 0.4068 | 0.7096 | 0.6697 | |
Nucleotide excision repair | 26 | 607 | 0.7382 | 0.2982 | ||
Other genes with suspected DNA repair function | 9 | 139 | 0.1382 | 0.3793 | 0.2542 | |
Other conserved DNA damage response genes | 9 | 127 | 0.8380 | 0.8285 | 0.4497 | |
Rad6 pathway | 5 | 54 | 0.6558 | 0.9324 | 0.8099 |
P values are based on the adaptive rank truncated product method.
Gene | Chromosome | # SNPs | (i) CIN3/CA | (ii) CIN3/CA | (iii) HPV persistence+ |
vs. RC | vs. HPV persistence | vs. RC | |||
12q24.2 | 36 | 0.0013 |
0.2537 | 0.1354 | |
6p21.3 | 15 | 0.0018 |
0.5355 | 0.0054 | |
8q13.3 | 77 | 0.0030 |
0.6935 | 0.0056 | |
15q15-q21.1 | 14 | 0.0038 |
0.2399 | 0.0612 | |
12q14 | 10 | 0.0038 |
0.0511 | 0.9136 | |
17q25.1 | 14 | 0.0038 |
0.5865 | 0.4742 | |
17q25.3 | 22 | 0.0081 | 0.0287 | 0.8114 | |
17q25.3 | 26 | 0.0098 | 0.0309 | 0.8457 | |
17q25.3 | 18 | 0.0094 | 0.0232 | 0.0864 |
FDR p≤0.2.
Results for all gene-based tests are shown in
Consistent with our gene/region-based analyses, we found evidence of an altered risk (approximately 2-fold) for CIN3/cancer for one or more SNPs in eight of the nine genes/regions with p-trends ≤0.001 (
Gene | rs # | (i) CIN3/CA | (ii) CIN3/CA | (iii) HPV persistence |
vs. RC | vs. HPV persistence | Vs. RC | ||
RS5757133 | 0.0007 | 0.1590 | 0.1114 | |
RS3784621 | 0.0004 |
0.1280 | 0.0262 | |
RS2894054 | 0.0001 |
0.8742 | 0.0002 | |
RS6926723 | 0.0004 | 0.6840 | 0.0034 | |
RS11177074 | 0.0003 | 0.0095 | 0.4011 | |
RS12307655 | 0.0005 |
0.2790 | 0.0034 | |
RS718802 | 0.0001 | 0.1844 | 0.0057 | |
RS12302655 | 0.00008 | 0.1909 | 0.0025 | |
RS17132382 | 0.0011 | 0.0103 | 0.5304 | |
RS4737999 | 0.0001 |
0.8918 | 0.0020 | |
RS4284050 | 0.0007 | 0.4994 | 0.0020 | |
RS10108002 | 0.0010 | 0.5160 | 0.0181 | |
RS9893818 | 0.0003 | 0.0019 | 0.8434 |
p<0.05 in oncogenic HPV+ restricted analyses.
SNP p values with FDR p<0.2 are denoted with astericks. Complete results for SNP-based results are shown in Supplemental Materials (
Results from haplotype-based analyses (defined by blocks of linkage disequilibrium) were generally consistent with the gene/region- and SNP-based findings. Haplotypes statistically significantly associated with CIN3/cancer included SNPs implicated in SNP-based analysis (as shown in
In this population-based study, we found nine genes/regions associated with CIN3/cancer, 6 of which remained significant at a FDR≤0.2 – three DNA repair genes (
Gene (Alias) | Chromosome | Gene Description |
Gene ontology** | Additional Comments | Selected References |
17q25.1 ?22q13.1 | Dosage suppressor of mck1 homolog, meiosis-specific homologous recombination | Female gamete generation; meiosis; reciprocal meiotic recombination; spermatogenesis; ATP binding; DNA binding | Rad51 homolog, meiosis | (6) | |
15q15-q21.1 | Deoxyuridine triphosphatase | DNA replication; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; dUTP disphosphatase activity | Modulation of nucleotide pools | (6) | |
17q25.3 | Hypothetical protein FLJ35220 | Editing/processing nucleases; incision 3′ of hypoxanthine and uracil | (6) | ||
6p21.3 | General transcription factor IIH, polypeptide 4 | DNA repair; nucleotide-excision repair, DNA damage removal; RNA elongation from RNA polymerase II promoter | Nucleotide excision repair; core TFIIH subunit p52 | (6) | |
4p16.2 | Polymerase (DNA directed) nu | DNA-dependent DNA replication; DNA-directed DNA polymerase activity | DNA polymerase; DNA crosslink repair | (6) | |
12q14 | Interferon, gamma | Cell cycle arrest; cell motion; signal tranduction | Cytokine with role in innate/adaptive immunity against viral/bacterial infections; role in tumor control | OMIM | |
12q24.2 | 2′-5′-oligoadenylate synthetase 1 | Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process | Role in resistance to viral infection via degradation of viral and cellular RNAs and impairment of viral replication. Also implicated in control of cell growth, differentiation, and apoptosis. | OMIM | |
12q24.2 | 2′-5′-oligoadenylate synthetase 2 | Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process | Resistance to viral infection (see OAS1) | OMIM | |
12q24.2 | 2′-5′-oligoadenylate synthetase 3 | Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process | Resistance to viral infection (see OAS1) | OMIM | |
8q13.3 | Sulfatase 1 | Heparin sulfate proteoglycan metabolic process; arylsulfatase activity | Coreceptor for heparin-binding growth factors and cytokines; involved in cell signaling | OMIM | |
17q25.3 | Transmembrane channel-like 8 | Epidermodysplasia verruciformis susceptibility gene | (20) | ||
17q25.3 | Transmembrane channel-like 6 | Epidermodysplasia verruciformis susceptibility gene | (20) |
As defined in NCBI Entrez Gene (
Results for our DNA repair pathway-based analyses were consistent with our individual SNP-based results. Specifically, we found that genes within nucleotide excision repair (which includes
Variants in two genes postulated to play a role in viral and HPV binding,
Three genes were associated primarily with progression to CIN3/cancer including
As described previously
In summary, our results require replication but build upon our previous report of potential host genetic variants relevant for HPV persistence and those relevant for progression to CIN3/cancer. If replicated, additional studies to pinpoint the causal SNP(s) and determine their biological relevance should be pursued. Future efforts should include further dissection of long-term persistence that likely leads to progression compared to short-term persistence that is less likely to progress to CIN3/cancer. The role of host gene and HPV methylation and their role on causal genes should also be considered. These data are important for future research in evaluating the interplay between viral and host genetics in determining risk of HPV persistence and for progression to CIN3/cancer.
The present study was nested within a population-based cohort study of women in Guanacaste, Costa Rica. Details of the cohort study methods
Women selected for the genetic study included: (i) all women in the cohort histologically confirmed with prevalent or incident CIN3 or cancer (n = 184); (ii) all women in the cohort with evidence of HPV persistence, defined as women positive for the same HPV type at two consecutive visits (n = 432) (median length of persistence: 25 months); and (iii) a random selection of controls from the cohort (n = 492)
The study was approved by both the US NCI and Costa Rica Institutional Review Boards and all subjects signed informed consent.
DNA was extracted from buffy coats with PureGene purification kits/Autopure protocol (Gentra Systems) at SeraCare (Frederick, MD). For the supplemental cases the DNA extraction was done at the University of Costa Rica using the same kit/protocol.
PCR-based HPV DNA testing with L1 MY09/MY11 consensus primer methods
Genotyping of tag SNPs from 305 candidate genes/regions (Genes and SNPs annotated in
Tag SNPs that failed manufacturing (ordered but did not convert), failed validation (no amplification or clustering) and assays that had less than 80% completion or 80% concordance with the 90 Hapmap CEU samples used for validation were excluded (n = 104). SNPs with low completion rate (<90% of samples) were further excluded (N = 138). SNPs with QC discordance among our 100 QC duplicates and among HapMap samples <98% were excluded (n = 383). We also excluded samples with a low completion rate (<90%) (N = 7). Hardy–Weinberg equilibrium was evaluated among controls. SNPs showing evidence of deviation from Hardy–Weinberg proportions (n = 49, p<0.0001) are denoted in
We evaluated a total of 416 women diagnosed with CIN3 or cancer, 356 women with HPV persistent infection, and 425 random controls for whom validated genotyping results were obtained.
We obtained pathway- and gene/region-based summary of associations using the adaptive combination of p-values
We calculated odds ratios (OR) and 95% confidence intervals (95% CI) for each genotype with each disease outcome, using the homozygous wild type (WT) genotype as the referent group. We first compared CIN3/cancer cases to random controls. We further evaluated whether the statistically significant associations for CIN3/cancer were consistent for HPV persistence and/or disease progression with the following respective comparisons: (i) HPV persisters compared to random controls and (ii) CIN3/cancer cases compared to HPV persisters.
We conducted both crude and analyses adjusted for age (<30, 30–49, 50+ years). For each outcome, we calculated the
We conducted haplotype analyses using two methods. First, we evaluated risk of cancer, progression and HPV persistence associated with haplotypes defined by SNPs within a sliding window of three loci across a gene (Haplo Stats, version 1.2.1, haplo.score.slide,
We sincerely thank Drs. Chris Buck and Patricia Day for their scientific contributions in suggesting candidate genes for evaluation with regard to their relevance in HPV binding. We are grateful to Sabrina Chen from the Information Management Services, Inc. (IMS) for data management and programming support. We are grateful to Amy Hutchinson and Belynda Hicks for their management of this genotyping effort at the NCI Core Genotyping Facility and for their scientific contributions to our research efforts. Results were presented in part at the 25th International Papillomavirus Conference, Malmo, Sweden, May 2009.
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