Abstract
BackgroundTo discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps.AimTo apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer.ResultsThe application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed in silico. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is CCNA1) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (SST, HTRA3 and NPTX1) are not methylated in normal cervix tissue.ConclusionThe application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown in silico and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.
Highlights
To discover cancer specific DNA methylation markers, large-scale screening methods are widely used
Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated
Gynaecological examination under general anaesthesia was performed in all cervical cancer patients for staging in accordance with the International Federation of Gynaecology and Obstetrics (FIGO) criteria [28]
Summary
To discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and reexpressed during functional reversal of DNA methylation upon treatment with demethylation agents. Such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. DNA methylation represents a modification of DNA by addition of a methyl group to a cytosine, referred to as the fifth base [1] This epigenetic change does not alter the primary DNA sequence and might contribute to overall genetic stability and maintenance of chromosomal integrity. Based on the sequence differences after bisulfite treatment, methylated DNA can be distinguished from unmethylated DNA, using methylation specific PCR (MSP) [6]
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