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Abstract Background Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies of the myeloid lineage. Its promoter region usually shows heterogeneous methylation and is only rarely fully methylated. The methylation status of CDKN2B can be used as a biomarker of response to treatment. Therefore the accurate characterisation of its methylation is desirable. Results MS-HRM was used to assess CDKN2B methylation in acute myeloid leukaemia (AML) samples. All the AML samples that were methylated at the CDKN2B promoter (40/93) showed varying degrees of heterogeneous methylation. Six representative samples were selected for further study. dMS-HRM was used to simultaneously count the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM. Conclusion dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B and other heterogeneously methylated genes. It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA. Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus. Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.

Background: MLH1 epimutation is characterised by constitutional monoallelic MLH1 promoter hypermethylation, which can cause colorectal cancer (CRC). Tumour molecular profiles of MLH1 epimutation CRCs were used to classify germline MLH1 promoter variants of uncertain significance and MLH1 methylated early-onset CRCs (EOCRCs). Genome-wide DNA methylation and somatic mutational profiles of tumours from two germline MLH1: c.-11C > T and one MLH1: c.-[28A > G; 7C > T] carriers and three MLH1 methylated EOCRCs (< 45 years) were compared with 38 reference CRCs. Methylation-sensitive droplet digital PCR (ddPCR) was used to detect mosaic MLH1 methylation in blood, normal mucosa and buccal DNA., Results: Genome-wide methylation-based Consensus Clustering identified four clusters where the tumour methylation profiles of germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs clustered with the constitutional MLH1 epimutation CRCs but not with the sporadic MLH1 methylated CRCs. Furthermore, monoallelic MLH1 methylation and APC promoter hypermethylation in tumour were observed in both MLH1 epimutation and germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs. Mosaic constitutional MLH1 methylation in MLH1: c.-11C > T carriers and 1 of 3 MLH1 methylated EOCRCs was identified by methylation-sensitive ddPCR., Conclusions: Mosaic MLH1 epimutation underlies the CRC aetiology in MLH1: c.-11C > T germline carriers and a subset of MLH1 methylated EOCRCs. Tumour profiling and ultra-sensitive ddPCR methylation testing can be used to identify mosaic MLH1 epimutation carriers., (© 2023. The Author(s).)

Closed-tube PCR methods (sometimes referred to as in-tube PCR methods) for locus-specific DNA -methylation analysis are methodologies in which the amplification and analysis of bisulphite-modified DNA take place in one tube without the need to remove the PCR products for further analysis. Closed-tube methodologies lend themselves to high-throughput applications and molecular diagnostics but are also applicable as a research tool. We review three closed-tube methodologies, methylation-sensitive high-resolution melting (MS-HRM), MethyLight, and sensitive melting after real-time analysis - methylation-specific PCR (SMART-MSP). Closed-tube detection can be performed by simultaneously amplifying both methylated and unmethylated templates and subsequent melting curve analysis (MS-HRM). Alternatively, methylation-specific primers are used in real-time quantitative PCR and monitored either by a fluorescent hydrolysis probe (MethyLight) or using a double-stranded DNA binding fluorescent dye with a subsequent quality control step by melting curve analysis (SMART-MSP).