Direct bisulphite conversion of cervical samples for DNA methylation analysis 113 5 INTRODUCTION Cervical cancer is the fourth most commonly occurring cancer in women 1. The usually slow progression from cervical high-risk human papillomavirus (HPV) infection to cancer via precancerous changes called cervical intraepithelial neoplasia (CIN), provides opportunities for prevention and early detection 1. Cytology-based screening has been a cornerstone of cervical cancer prevention strategies for decades. In recent years, HPV-based screening has been adopted in several countries given a better protection against cervical cancer and precancer than cytology 2. With the implementation of HPVbased screening, triage testing has become important to increase specificity and positive predictive value, while retaining accurate identification of women with high-grade CIN, who require follow-up management. Epigenetic biomarkers have a strong potential to be implemented as molecular triage tool. At present, the most widely studied epigenetic alteration is the methylation of DNA at CpG dinucleotides (5-methylcytosine), which are usually highly concentrated in CpG islands within the promoter regions of human genes 3. Studies have reported a gradual increase in DNA methylation of specific hostcell genes with higher grade of CIN, reaching highest levels in cervical cancer 4-6. A wellstudied methylation marker panel with host-cell genes FAM19A4 (currently known as TAFA4) and miR124-2 showed good triage performance in HPV-positive women 7-9, as recently validated in a large cross-sectional, multicentre European cohort study across four different countries 10. A commonly used approach for DNA methylation analysis is genomic DNA isolation from a clinical specimen followed by sodium bisulphite treatment prior to PCR amplification. Treating DNA with sodium bisulphite converts unmethylated cytosines to uracil while methylated cytosines remain unchanged. This conversion thus generates specific changes in the DNA sequence that depend on the methylation status of the individual cytosines and can be measured by subsequent quantitative methylation-specific PCR (qMSP) 11, 12. This protocol could be made more efficient by bisulphite conversion directly on cells, without the need for prior genomic DNA isolation and normalisation of DNA input. Here, we evaluated a direct cell conversion protocol on a series of clinician-collected cervical samples. We compared results of ACTB control gene and methylation of FAM19A4 and miR124-2 genes on bisulphite-treated DNA derived from direct cell conversion to those from a protocol involving prior DNA isolation and normalisation (reference protocol). Direct cell conversion protocols could further improve efficiency and considerably enhance the practicability and operations of methylation analysis in
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