Our key findings in this case-control study of Tanzanian women are that after adjusting for HPV infection, age, OC use, and HIV-1 status, a 5% increase in DNA methylation at the PEG3 DMR was associated with a 1.6 fold increase in ICC risk. We also found that PEG3 DMR hypermethylation was correlated with HPV infection; a correlation that was stronger for high risk as compared to low risk HPV infection. As would be expected, HPV infection was associated with increased risk of CIN1/2/3 and ICC. We present the first evidence in support of the hypothesis that aberrant methylation of the PEG3 DMR is an important co-factor in the development of ICC, especially among women infected with HR HPV.
Our data suggests that increasing grade of lesion from CIN to ICC correlates with HPV infection and PEG3
DMR hypermethylation and is consistent with DNA methylation-mediated repression of PEG3
as found in previous studies. Hypermethylation of various genes (i.e. MGMT
, and MHL1
) in ICC case control sudies has been reported 
. Previous studies have reported DNA methylation changes and HPV status in head and neck squamous cell cancer 
. These findings support the idea that the presence of aberrantly methylated genes could be used as a relatively sensitive and specific screening assay to detect CIN and ICC. These previous studies did not investigate PEG3
. To our knowledge this is the first study done with a human population that investigated the relationship between PEG3
DMR status and HPV infection and how this plays a role in CIN and ICC. Although cause and effect cannot be established in this case-control study, our findings suggest that PEG3
DMR methylation is a potential mechanism by which susceptibility to progression to ICC may occur, and thus may be a useful marker to identify CIN cases likely to progress.
The mechanisms by which PEG3
DNA methylation increases risk of ICC are unclear. However, there is evidence suggesting that PEG3
plays an important biological role in p53/c-myc mediated apoptosis, implicating PEG3
as a gene whose function may be in part to prevent carcinogenesis 
. The p53-mediated apoptosis pathway has two potential outcomes: induction of a) growth arrest or b) cell death; Peg3 has been shown to play a role downstream of p53 activating apoptosis via its interaction with Bax. Peg3 interacts with Bax, resulting in apoptosis 
. These prior reports, together with our findings, support the hypothesis that PEG3
functions as an important tumor suppressor in carcinogenesis.
The association found here is consistent with findings from in vitro
and in vivo
studies showing that the PEG3
promoter is hypermethylated with consequent transcriptional repression in ovarian and endometrial cancers 
. In cervix, ovarian, and endometrial cancer cell lines PEG3
is silenced suggesting that during carcinogenesis, hypermethylation may be selected for in order to inhibit the pro-apoptotic function of PEG3. Our case control study shows an association between hypermethylation of PEG3
and ICC but not CIN, suggesting that these methylation alterations take place during transformation rather than in pre-cancerous lesions. Alternatively, the attenuation in risk may be due to combining low grade CIN largely comprised of lesions likely to regress, with higher-grade CIN cases, the majority of which have potential to progress and become ICC. Intriguingly, the correlation of HPV infection, an etiologic agent of ICC, and PEG3
hypermethylation is consistent with a multi-step process that starts with epigenetic mechanisms and HPV infection.
The main limitation of this study is the small sample size to examine PEG3
DMR methylation in relation to grade specific CIN, after accounting for the effect of HPV infection. It is possible that our inability to find associations between PEG3
methylation and CIN was due to combining CIN (in whom the majority or women are likely to regress) and CIN2 and CIN3 (in whom a smaller proportion persist or progress) 
. However, we had adequate statistical power to evaluate PEG3
and ICC risk. Another limitation is the case-control design, limiting our ability to infer PEG3
methylation as an important factor in progression. However, identifying methylation marks associated with case-control differences is a necessary step allowing for examination of this marker in longitudinal studies currently under way by several groups 
Despite these limitations, we found hypermethylation of the PEG3 DMR increased the risk for ICC after adjusting for known confounders. We also found a strong correlation between HPV genotype and DNA methylation at the PEG3 DMR. Cytosine methylation is a stable modification in human tissue samples, and therefore PEG3 DMR methylation status could potentially be used as a marker to identify CIN likely to progress to ICC. Larger studies in a more diverse study population are required to replicate these findings.