Base5, a versatile, highly integrated high-throughput methylation profiling platform for methylation specific PCR based marker identification applied to colorectal cancer

A61 Testing the methylation status of one or more genomic loci is a new principle applicable to diagnosis and prognosis of disease, as well as prediction of therapeutic response. In an attempt to increase the success rate of marker discovery work in the field of methylation a new integrated system including bioinformatics, high throughput marker testing and data analysis has been created. The newly devised ‘Base5’ system is described and proof of principle data from studies in the field of colorectal cancer are presented. Materials and Methods: The Base5 methylation profiling platform (Base5) currently comprises several different modules: The first module is a set of bioinformatics tools which allow the design of Methylation Specific PCR (MSP) assays in a highly efficient way, based on any annotated or known genomic DNA sequence. With this tool 224 different MSP assays in the promoter region of 145 different genes have been newly designed. In addition to these new candidates, several already known methylation markers for colorectal cancer were included in a “design list”. This list was used for manufacturing high throughput arrays capable of analyzing 12 different samples in parallel with all the assays of interest. The second module of the Base5 system, a high throughput real time detection system was used to determine the methylation status of the genes processed in module 1. The equivalent of 200ng of genomic, bisulphite converted DNA from 90 samples (60 colorectal cancer samples and 30 normal colorectal tissue samples) was tested for the presence of methylated alleles. The third module of the Base5 system was used to analyze and present the results in “methylation tables” which indicate the methylation status of each sample tested for each of the assays. Using the methylation tables, sensitivity and specificity for each gene was calculated and genes were ranked by specificity and sensitivity. In a final step, the performance of known colorectal cancer markers (CHFR, MGMT and others) included in the design was compared to the new candidate markers screened with the system. Results: Using the Base5 system it was possible to design 224 different MSP assays within two workdays. A total of 20160 MSP assays was performed by one technical staff within four workdays including genomic DNA isolation, bisulphite conversion, real-time detection and data interpretation. Known methylation markers of colorectal cancer included in the list of designs were confirmed among the best performing assays. New markers, so far not associated with colorectal cancer, could be determined which perform as well or even better than the known markers, with a relatively small effort.