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 (GTF2H4, DUT, and DMC1) and three viral infection and cell entry related genes (OAS3, SULF1, and IFNG). A unique aspect of our study is the ability to separately evaluate associations with the two important transition steps in the natural history of cervical cancer – viral persistence and progression to precancer/cancer. Of the regions/genes found to be important, the association was primarily with HPV persistence for GTF2H4 and SULF1. IFNG and the epidermodysplasia verruciformis (EV)-associated genes (TMC6-EVER1 and TMC8-EVER2) were primarily associated with progression to CIN3/cancer. All top genes derived from the gene/region- and SNP-based analyses are annotated and briefly described in .
Description of top-ranked genes.
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 GTF2H4
) and modulation of nucleotide pools (which includes DUT
) to be associated with HPV persistence. The DNA repair family of editing/processing of nucleases was associated with progression to CIN3/cancer and though several genes were statistically significant, none were notable at FDR < 0.2. The association between the Fanconi Anemia family of genes with HPV persistence is also consistent with our previous analyses that reported FANCA
variants associated with HPV persistence (5). There have been no reports of polymorphisms in GTF2H4
with HPV persistence, cervical cancer, or any other cancer to date. However, GTF2H4
is located on chromosome 6p21.3 within the HLA
region and is thus of note as a number of studies have investigated HLA
Class II and I genes with cervical cancer and have consistently identified alleles (e.g., HLA
*1301) associated with cervical cancer 
. Whether the observed association between GTF2H4
variants and HPV persistence is due to its biological function and its capacity as a DNA repair gene or to potential linkage disequilibrium with HLA
requires further evaluation.
Variants in two genes postulated to play a role in viral and HPV binding, OAS3
, were also associated with HPV persistence in our population. OAS3
plays a role in resistance to viral infection via degradation of viral and cellular RNAs and impairment of viral replication. Specifically, the OAS family of genes (OAS1, OAS2, OAS3
) is induced by interferon. When enzymatically active OAS are bound to viral RNA, RNase L is activated, resulting in the degradation of viral RNA 
. Notably, OAS1
SNPs were also associated with HPV persistence in our population (). SULF1
(sulfatase 1) is involved in cell signaling and is a coreceptor for heparin-binding growth factors and cytokines. Sulfs potentially play a role in a cellular feed-back mechanism where they edit the sulfation of multiple heparin sulfate proteoglycans 
. This is of particular interest as heparin sulfate proteoglycans are thought to be the primary attachment factor for HPV and treatment with heparin and heparin sulfate have inhibited infection of some HPV types 
Three genes were associated primarily with progression to CIN3/cancer including IFNG
, a cytokine that plays a role in innate immunity against viral or bacterial infections. It also induces the OAS family of genes which leads to degradation of viral RNA. We also report an association between genes in EVER1/TMC6
and a SNP that lies between the two genes with progression to CIN3/cancer. Mutations in either EVER1
genes are well-documented in the rare skin carcinoma, epidermodysplasia verruciformis (EV), which is characterized by infection with HPV5 
. That common polymorphisms within EVER1
are also associated with progression to CIN3/cancer may potentially suggest a larger role, though modest, for EVER1
genes in HPV susceptibility and subsequent risk for disease.
As described previously 
, study limitations may include potential survival bias as the supplemental cases identified in Guanacaste were retrospectively ascertained and DNA was not obtained for deceased cases. Another limitation is our inability to evaluate invasive cervical cancer separately. Future studies with larger numbers of cases will be needed to address whether specific genes are involved in the transition from in situ to invasive disease. Although we targeted a priori
genes and used a FDR of <0.2 to determine which genes were notable, we cannot exclude the possibility of false positives (or false negatives). Importantly, because our population comprises Costa Ricans and genes were tagged based on Caucasian and Yoruban populations where data are available from HapMap, it is plausible that gene coverage in our Costa Rican population is incomplete. Study strengths include our population-based study design. This design approximates a case-cohort design since the proportion of women in the cohort with CIN3 or cancer is small. Another study strength is our ability to evaluate genes relevant for HPV persistence and genes relevant for disease progression. Further, our study had rigorous follow-up, HPV testing and pathology review for case definitions. Our tag-SNP approach also allowed for broader coverage of each a priori
gene/region investigation, compared to prior candidate SNP-based genotyping approaches. Our evaluation of the DNA repair pathway was near complete based on most recent literature and allowed for pathway-based analyses. However, our viral binding and cell entry genes were not amenable to similar analyses. We acknowledge that our a priori
approach sampled a small subset of genes in the genome and we therefore likely missed other important associations. Increased power and replication of our results are required and larger efforts in genome-wide association studies should shed additional light on genes and regions of significance for cervical cancer. Our study, however, remains the only one designed to delineate between associations with HPV persistence and progression to CIN3/cancer.
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.