Methylation-Specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences

Abstract

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA–probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA–probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader–Willy syndrome, Angelman syndrome or acute myeloid leukemia.

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

“…The MS-MLPA was performed as published previously (Nygren et al, 2005), using two probe mixtures (P041A and P041B). In total, the probe mixtures contained a panel of probes specific for 29 candidate tumour suppressor genes ( Table 1).…”

“…The percentage of methylation was calculated by dividing normalised peaks in the Hha1-digested reaction by the normalised peaks in the undigested control reaction. Methylation below the threshold level of 10% was considered background (Nygren et al, 2005). All samples were analysed twice and scored positive for methylation when in both experiments the percentage of methylation for each individual probe was above the threshold.…”

“…Notably, all eight genes that were frequently methylated in the HPV-transfected cell lines and cervical cancer cell lines (i.e. TP73, ESR1, RARb, DAPK1, MGMT, CADM1, CDH13 Figure 1 Overview of the MS-MLPA procedure (Nygren et al, 2005). The gene-specific probes spanning a recognition site for the restriction enzyme Hha1 are hybridised to the target DNA and subsequently ligated and digested with the methylation-sensitive enzyme Hha1.…”

“…MS-MLPA is a PCR-based technique allowing the semiquantitative detection of changes in DNA promoter methylation of multiple genes in a single reaction (Nygren et al, 2005). Discrimination between methylated and unmethylated targets is based on the annealing of probes containing a recognition site for the methylation-sensitive restriction enzyme HhaI.…”

Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in oligodendroglial tumors

“…Methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA) is a polymerase chain reaction (PCR)‐based technique that allows for identification of the methylation status of multiple genes in a single experiment 10. Based on the annealing of probes containing a recognition site for the methylation‐sensitive restriction enzyme Hha I, this technique has been applied in several studies of cancer 11.…”