General Information: The SALSA MS-MLPA Probemix ME011 MMR is a research use only (RUO) assay for the detection of aberrant methylation of one or more sequences of the MLH1, MSH2, PMS2 and MSH6 genes, and the presence of the BRAF c.1799T>A; p.Val600Glu point mutation to determine a sporadic nature of Lynch syndrome-related cancer in tumour tissue. This probemix can also be used to detect deletions/duplications in the promoter regions of the aforementioned genes and the EPCAM 3’ region to determine whether follow-up germline Lynch syndrome testing should be considered.
CpG-islands are located in or near the promoter region or other regulatory regions of approximately 50% of human genes. Aberrant methylation of CpG-islands has been shown to be associated with transcriptional inactivation of genes in a wide spectrum of human cancers. The genes mentioned above are frequently silenced by methylation in tumours, but are unmethylated in blood-derived DNA, with the exception of constitutional epimutations.
The Mismatch Repair (MMR) system is critical for the maintenance of genomic stability. Defects in the cell’s MMR system may lead to the accumulation of mutations resulting in the initiation of cancer. Several MMR genes are involved in Lynch syndrome (formerly known as hereditary nonpolyposis colon cancer (HNPCC)). Genetic alterations in the MLH1 and MSH2 genes have been found in up to 90% of Lynch syndrome cases. Genetic alterations in MSH6 and PMS2 genes are less frequently detected in Lynch syndrome patients. Around 1-3% of Lynch syndrome cases are explained by EPCAM deletions. Elimination of the EPCAM transcription termination signal results in transcription continuing into MSH2 and silencing of the MSH2 promoter by methylation.
In sporadic colon cancer, hypermethylation is a more frequent mechanism than point mutations or copy number alterations for transcriptional silencing of the MLH1 gene. The probes targeting GCGC sites in the C and D “Deng” region of the MLH1 gene are of main interest (Deng et al. 1999). In addition, the BRAF p.Val600Glu mutation is a frequently found pathogenic variant in sporadic colorectal cancers. Hypermethylation of the MLH1 promoter or the BRAF p.Val600Glu mutation are rare in tumours of Lynch syndrome patients.
Of note, constitutional inactivation of MLH1 by methylation, together with a somatic mutation in the functional allele, has been reported as a rare cause of Lynch syndrome and may in rare cases be a heritable disease mechanism (Goel et al. 2011, Morak et al. 2018).
Promoter inactivation by methylation of MSH6 or PMS2 has not been reported according to our literature review in Lynch syndrome patients or described as somatic cause in colorectal or endometrial tumours.
More information is available at
https://www.ncbi.nlm.nih.gov/books/NBK1211/
Probemix content: The SALSA MS-MLPA Probemix ME011-C1 MMR contains 37 (MS-)MLPA probes with amplification products between 123 and 454 nt. Fourteen MS-MLPA probes contain a HhaI recognition site and provide information on the methylation status of MLH1, MSH2, MSH6 and PMS2. All probes present, including three EPCAM probes, will also give information on copy number changes in the analysed sample. Furthermore, the probemix also contains a probe specific for the BRAF p.Val600Glu mutation, which will only generate a signal when the mutation is present. In addition, 17 reference probes are included which are not affected by HhaI digestion and target relatively quiet regions in colorectal tumours. Also, two digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete. Complete probe sequences and the identity of the genes detected by the reference probes is available in table 2d and online (www.mlpa.com).
This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity Fragments (Q-fragments), two DNA Denaturation Fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment (see table below). More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mlpa.com.
SALSA Binning DNA SD029: The SD029 Binning DNA provided with this probemix can be used as Binning DNA sample for binning of one mutation-specific probe (226 nt probe 08780-SP0039-L08904 BRAF p.Val600Glu mutation). SD029 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD029 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signal(s), as for this purpose true mutation/SNP positive patient samples or cell lines should be used. It is strongly advised to use DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For further details, please consult the SD029 Binning DNA product description provided.
This product is for research use only (RUO).
sample DNA
Sample DNA developed for this product: