Novel epigenetic changes in CDKN2A are associated with progression of cervical intraepithelial neoplasia

Abstract

Objective To conduct a comprehensive mapping of the genomic DNA methylation in CDKN2A, which codes for the p16INK4A and p14ARF proteins, and 14 of the most promising DNA methylation marker candidates previously reported to be associated with progression of low-grade cervical intraepithelial neoplasia (CIN1) to cervical cancer. Methods We analyzed DNA methylation in 68 HIV-seropositive and negative women with incident CIN1, CIN2, CIN3 and invasive cervical cancer, assaying 120 CpG dinucleotide sites spanning APC, CDH1, CDH13, CDKN2A, CDKN2B, DAPK1, FHIT, GSTP1, HIC1, MGMT, MLH1, RARB, RASSF1, TERT and TIMP3 using the Illumina Infinium array. Validation was performed using high resolution mapping of the target genes with HELP-tagging for 286 CpGs, followed by fine mapping of candidate genes with targeted bisulfite sequencing. We assessed for statistical differences in DNA methylation levels for each CpG loci assayed using univariate and multivariate methods correcting for multiple comparisons. Results In our discovery sample set, we identified dose dependent differences in DNA methylation with grade of disease in CDKN2A, APC, MGMT, MLH1 and HIC1, whereas single CpG locus differences between CIN2/3 and cancer groups were seen for CDH13, DAPK1 and TERT. Only those CpGs in the gene body of CDKN2A showed a monotonic increase in methylation between persistent CIN1, CIN2, CIN3 and cancers. Conclusion Our data suggests a novel link between early cervical disease progression and DNA methylation in a region downstream of the CDKN2A transcription start site that may lead to increased p16INK4A/p14ARF expression prior to development of malignant disease.

New Insight into microRNA Functions in Cancer: Oncogene–microRNA–Tumor Suppressor Gene Network

“…also induce dysfunction of oncogene and TSG, which are observed in kinds of cancers (Grønbæk et al, 2007; Singh et al, 2016; Wijetunga et al, 2016). Many studies showed that the collaboration of epigenetic and genetic changes is critically vital to drive the cancer development and progression (Baylin and Jones, 2016).…”

Molecular tests potentially improving HPV screening and genotyping for cervical cancer prevention

“…This is an expanding area where global cellular and/or viral changes including hypomethylation and hypermethylation are being investigated as diagnostic tools for detection of cervical precancerous lesions [47,78–81]. Determining methylation levels on HR-HPV genomes has the potential to detect precancerous lesions with higher specificity than HPV testing alone [26].…”

“…Ongoing efforts will continue to require large prospective clinical studies to validate sensitivity, specificity, positive and negative predictive values for new techniques such as NGS that have been shown to be accurate and are becoming more cost-effective, where computational advances allow for increased sensitivity and depth of sequencing [83]. Further research on how changes in the viral and host methylome are associated with cervical cancer development should provide mechanistic insights facilitating prevention and treatment [74,81]. …”

“…Nonetheless, there is potential for use of methylation as a reflex panel of tests (viral and/or cellular) that would provide improved risk stratification for triage of women with HR-HPV infections [80,87]. As an example, there is increasing evidence towards CDKN2A (cyclin-dependent kinase inhibitor 2A, gene encoding p16) as a “candidate” showing differential levels of methylation between low-grade CIN and high-grade CIN [81]. These considerations still require proper evaluation and validation for future applications.…”

“…Sequencing of small fragments of bisulfite-converted HPV DNA using NGS platforms provides highly informative quantitative measurements of single CpG site methylation levels that are associated with disease status [81,83,87]. The extension of this association to other HR-HPV types looks promising, but it has been difficult to study due to the lower prevalence of HR-HPV types other than HPV16 [88–90].…”