Title : Methylation-sensitive high resolution melting (MS-HRM): A sensitive and specific method for methylation assessment in human disease
DNA methylation is an epigenetic mechanism which implies heritable changes of gene expression without a change in the primary DNA sequence. Covalent histone modifications and methylation changes of cytosine at CpG dinucleotides are the most widely investigated epigenetic mechanisms. DNA regions with a relatively high CpG dinucleotide content are referred to as CpG islands that are distributed in a non-random manner across the human genome and often span 5’ untranslated region (UTR), 3’ UTR, promoter region and the first exon of protein coding genes. Methylation of CpG islands usually acts to turn off (silence) transcription by recruiting histone deacetylases thereby inducing the formation of inactive chromatin. Mapping of methylation patterns in CpG sites is an important tool for understanding both normal and pathogenic gene expression events.
Numerous technique are used for detection of CpG methylation, among them, PCR-based protocols are most widely used. Because PCR amplification removes methylation marks, the DNA template is chemically modified by sodium bisulfate that converts all unmethylated cytosines to uracil, leaving methylated cytosines unaltered and preserving methylation information before PCR amplification.
Subsequent amplification of bisulfite-modified template results in different amplicons from methylated and unmethylated templates with different melting profiles when subjected to thermal denaturation.
The methylation-sensitive high resolution melting (MS-HRM) technology is based on the comparison of the melting profiles of sequences that differ in base nucleotide composition. The PCR product originating from the methylated allele will have different GC content from PCR product derived from unmethylated variant of the same locus. MS-HRM allows for estimation of the methylation level by comparing the melting profiles of unknown PCR products to the melting profiles of PCR products derived from standards with a known unmethylated to methylated template ratio (range from 0 to 100 methylation percentage).
Here, we show the application of MS-HRM in three different studies:
1) detection of methylation levels in the 3’UTR of dystrophia myotonica protein kinase (DMPK) gene in a cohort of 66 myotonic dystrophy type 1 (DM1) patients (age 38.6±12.5 years) and 30 age-matched healthy controls (age 40.3±13.8 years) [Santoro et al., Biochim Biophys Acta. 2015;1852:2645-52. doi: 10.1016/j.bbadis.2015.09.007].
2) detection of promoter methylation levels in cannabinoid type 1 (CNR1) and 2 (CNR2) receptors of 12 multiple sclerosis secondary progressive (MSS-SP) patients (age 54.2±11.7 years) before and after treatment with Sativex®.
[Santoro et al., J Neurol Sci. 2017;379:298-303. doi: 10.1016/j.jns.2017.06.017].
3) Detection of the OCT4 promoter methylation in human induced pluripotent stem cells of monogenic diseases.
[Spitalieri et al., Cell Reprogram;17:275-87. doi: 10.1089/cell.2015.0003].
The MS-HRM protocol provides a high-throughput platform for cost- and labor-efficient screening for methylation changes. Moreover, the simplicity and high reproducibility of this technique makes MS-HRM a possible method of choice for methylation assessment in both fundamental research and research in regards to possible diagnostic applications.
Presentation Learning Outcome
- MS-HRM protocol provides a high-throughput platform for cost- and labor-efficient screening for methylation changes.
- The simplicity and high reproducibility of this technique makes MS-HRM a method of choice for methylation assessment in both fundamental research and research in regards to possible diagnostic applications.
- Limitations: the melting analyses do not allow detailed information about the methylation of single cytosines within the sequence of interest to be obtained. These can be assessed only by DNA sequencing technologies.