Intended use: This SALSA MLPA probemix ME011 Mismatch Repair Genes is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² to determine methylation status of selected GCGC sites in the promoter regions of MLH1, MSH2, PMS2 and MSH6 genes and to detect the BRAF p.V600E (=c.1799T>A) point mutation in colorectal and endometrial cancer, in order to stratify the risk of having Lynch syndrome and identify cases which might benefit from subsequent germline genetic testing. In addition, this assay can be used to detect deletions or duplications in the 3' region of the EPCAM gene and the promoter regions of MLH1, MSH2, PMS2 and MSH6 genes in order to aid diagnosis of Lynch syndrome.

This assay is for use with peripheral blood derived genomic DNA and also for DNA derived from fresh, frozen and FFPE tissue of colorectal and endometrial cancer, given that tumour cell content of the patient sample is higher than 50%. For analysis of tumour samples, same or similar source of tissue, identical sample treatment, identical DNA extraction method and a minimum of three healthy reference samples should be used. Methylation status, deletions and duplications and the mutation status of BRAF p.V600E should be verified by another technique. Copy number changes detected by only a single probe always require validation by another method.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

¹Please note that this probemix is for In Vitro Diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO).
²To be used in combination with a SALSA MLPA Reagent Kit, Coffalyser.Net analysis software, SALSA HhaI, and SD086 Binning DNA.

Clinical background: The main genes in the DNA mismatch repair (MMR) system are MLH1, PMS2, MSH2 and MSH6. Heterodimers of proteins encoded by these genes (MLH1/PMS2 and MSH2/MSH6) repair and prevent DNA mutations. Defects in the cell’s MMR system may lead to the accumulation of mutations resulting in the initiation of cancer. Epigenetic silencing or loss of function mutations in the above-mentioned genes cause MMR deficiency and microsatellite instability (MSI). Heterozygous germline mutations in any of the MMR genes result in Lynch syndrome (LS, also known as hereditary nonpolyposis colorectal cancer, or HNPCC) - an autosomal dominant cancer predisposition condition. LS is characterised by an increased risk of colorectal cancer (life time risk 52-82%), endometrial cancer (life time risk 25-60%), gastric cancer (life-time risk 6%-13%), ovarian cancer (life time risk 4-12%) and other cancers such as hepatobiliary tract, urinary tract, brain and skin. Genetic alterations in the MLH1 and MSH2 genes have been found in up to 90% of LS cases, whereas MSH6 and PMS2 gene alterations are less frequently detected. Around 1-3% of LS 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. More information on LS is available at https://www.ncbi.nlm.nih.gov/books/NBK1211/.

In sporadic MSI-positive colorectal and endometrial carcinomas the MLH1 promoter is methylated in 10-20% of cases resulting in the loss of MLH1 expression (Herman et al. 1998, Cunningham et al. 1998, Kane et al. 1998, Esteller et al. 1998, Simpkins et al. 1999). MLH1 promoter methylation testing is performed in order to differentiate between sporadic MSI and LS. The CpG sites in the C- and D- “Deng” regions of the MLH1 gene are of main interest (Deng et al. 1999). MLH1 promoter methylation analysis on tumor tissue can improve the selection of patients for LS genetic testing and thus provide substantial cost reductions (Perez-Carbonell et al. 2010). Of note, in recent years rare cases of constitutional MLH1 hypermethylation together with a somatic mutation in the functional allele have been reported in LS families (Pinto et al. 2018, 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 LS patients or described as a somatic cause in colorectal or endometrial tumours.

BRAF pathogenic variants, most commonly the p.V600E mutation, are another important molecular marker identified in ~15% of sporadic colorectal cancers (Bouzourene et al. 2010). These mutations have a strong association with MLH1 promoter methylation, and therefore BRAF mutational and MLH1 methylation tests are often performed concurrently. BRAF mutation is frequently present in sporadic colorectal cancer with methylated MLH1, but not in LS. Given the comparative rarity of BRAF mutation and MLH1 hypermethylation in LS tumours, the testing of those is done in tumour tissue of colon cancer cases to differentiate LSassociated cancer from more common sporadic cancers. BRAF pathogenic variants, however, are not common in sporadic endometrial cancers; thus, BRAF testing is not helpful in distinguishing endometrial cancers that are sporadic from those that are LS-related.

Probemix content: The SALSA MS-MLPA Probemix ME011-D1 Mismatch Repair Genes contains 34 (MS-)MLPA probes with amplification products between 123 and 398 nucleotides (nt). 14 MS-MLPA probes contain an HhaI recognition site and provide information on the methylation status of selected GCGC sites in the promoter regions of MLH1, MSH2, MSH6 and PMS2 genes. 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.V600E mutation and a probe specific for the rs104894994 single nucleotide polymorphism (SNP), which will only generate a signal when respectively the mutation or the SNP are present. In addition, 13 reference probes are included that are not affected by HhaI digestion and target relatively copy number stable regions in colorectal and endometrial cancer. Also, two digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete. The identity of the genes detected by the reference and digestion control probes are available in Table 2b of the product description 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. More information on how to interpret observations on these control fragments can be found in the MS-MLPA General Protocol and online at www.mlpa.com.

SALSA Binning DNA SD086: The SD086 Binning DNA provided with this probemix can be used for binning of one mutation- and one SNP-specific probe (226 nt probe 08780-SP0039-L08904 BRAF p.V600E mutation and 289 nt probe 22572-L31773 MLH1 rs104894994 (C>T) SNP probe). SD086 Binning DNA is a mixture of genomic DNA from healthy individuals and synthetic DNA that contains the target sequence detected by the above mentioned probes. Inclusion of one reaction with 5 μl SD086 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, SD086 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 or SNP signal. It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD086 Binning DNA product description, available online: www.mlpa.com.

Sample DNA
Sample DNA developed for this product:

• SD086 Binning DNA - included with this probemix.