General introduction and thesis outline 27 1 HPV16E6E7-transduced primary foreskin keratinocytes and is associated with disease progression 128. Downregulation of miR124-2 was identified through genome-wide studies on miRNA expression and has been linked to increased promoter methylation in cervical cancer 129. ASCL1, LHX8 and ST6GALNAC5 were discovered through an array-based methylation discovery screen (Infinium 450K array) on hrHPV-positive self-collected samples 126, while methylation markers GHSR, SST and ZIC1 were identified through a methyl-binding domain-enriched DNA-based discovery screen (MBD-seq) on high-risk HPV-transformed cell lines and cervical tissue specimens 127. Methylation levels of these genes increase progressively with the severity of underlying disease and are significantly higher in CIN3 and cervical cancer lesions compared to control women without evidence of CIN2+ 123, 126, 127. 1.4.2 DNA METHYLATION MARKER ANALYSIS For validation studies and diagnostic purposes, a targeted and more sensitive method to detect DNA methylation is preferred over genome-wide techniques. Multiplex quantitative methylation-specific PCR (qMSP) is an efficient and reproducible technique that can detect multiple methylated genes and a reference gene in a single assay. This method enables multiple methylation targets to be analysed using a single sample aliquot, which can reduce target-to-target variations and allow for the monitoring of sample adequacy for PCR purposes by an internal reference gene 130. Additionally, this saves material, time, and costs while improving quality control. The method can also be automated, making it suitable for high-throughput settings. The qMSP technique starts with bisulphite treatment of DNA to convert cytosine to uracil, while leaving methylated cytosines unchanged. The converted DNA is then amplified by PCR using specific primer pairs for methylated DNA and fluorescent probes, with methylation status quantified by fluorescence monitoring. 1.4.3 CLINICAL IMPLICATIONS OF DNA METHYLATION ANALYSIS In recent years, the use of DNA methylation analysis of host-cell genes as a clinical tool for detecting cervical cancer and precancerous lesions has gained considerable attention 131. Analysis of DNA methylation can be performed on different sample types, including clinician-collected cervical samples, self-collected samples, cervical tissue specimens and even urine. Methylation assays can be used for several purposes 132, 133: 1. As primary triage of hrHPV-positive women to detect CIN3+; 2. As secondary triage for women with minor cytological abnormalities to identify those with the highest risk of CIN3+;
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