The SALSA MS-MLPA probemix ME011 Mismatch Repair Genes is an in vitro diagnostic (IVD) semi-quantitative assay to determine methylation status of selected GCGC sites in the promoter regions of the human MLH1, MSH2, PMS2 and MSH6 genes and to detect the BRAF p.V600E (c.1799T>A) point mutation in order to stratify the risk of having Lynch syndrome. In addition, this assay can be used for the detection of deletions or duplications in the human 3' region of the EPCAM gene and the promoter regions of MLH1, MSH2, PMS2 and MSH6 genes in order to confirm a potential cause for and clinical diagnosis of Lynch syndrome and at-risk family members.

For the full intended purpose, see the product description.

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, endometrial cancer, gastric cancer, ovarian cancer 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 (Kuiper et al. 2011, Ligtenberg et al. 2009, Niessen et al. 2009). 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 (Cunningham et al. 1998, Esteller et al. 1998, Herman et al. 1998, Kane et al. 1997, 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 (Goel et al. 2011, Morak et al. 2018, Pinto 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, is 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 mutation 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 LS-associated 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.

SALSA MLPA Probemix ME011 Mismatch Repair Genes is CE-marked for in vitro diagnostic (IVD) use.

This assay is for research use only (RUO) in all other territories.

SALSA Binning DNA SD086 is an artificial DNA sample with a signal for all probes in the ME011 Mismatch Repair Genes probemix. Inclusion of a reaction with SD086 in initial experiments and in experiments following a change in electrophoresis conditions is recommended to aid in the creation of a bin set that links peaks to the probes that produce them. Binning DNA cannot be used as a reference sample in the MLPA data analysis, and cannot be used to quantify the signals of mutation-specific probes.

A vial of SALSA Binning DNA SD086 is included with every order of the ME011 Mismatch Repair Genes probemix, but it is possible to order additional vials separately.

For more information, see the product description.

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below have been tested with the current (D1) version of this product and have been shown to produce useful results.

Chromosome 2

• Coriell NA10401: Heterozygous duplication affecting the probes for EPCAM, MSH2 and MSH6, the digestion control probe at 132 nt and the reference probes at 127 and 190 nt. All MMR genes are not methylated.
• Coriell NA13451: Heterozygous deletion affecting the probes for EPCAM, MSH2 and MSH6. All MMR genes are not methylated.

Chromosome 3

• Coriell NA04127: Heterozygous duplication affecting the probes for MLH1. All MMR genes are not methylated.
• DSMZ ACC-427 (DU-4475): Positive for the BRAF c.1799T>A (p.V600E) mutation. Moderate methylation (~50%) for 247 and 278 nt MLH1 probe targets, all other genes are not methylated.
• DSMZ ACC-203 (SK-N-MC): Heterozygous deletion affecting the probes for MLH1, the digestion control probe at 132 nt and the reference probes at 178 and 398 nt. Low methylation (10-15%) for all MLH1 probe targets, all other genes are not methylated.

Chromosome 7

• Coriell NA07081: Heterozygous duplication affecting the probes for PMS2. All MMR genes are not methylated.
• DSMZ ACC-277 (DK-MG): Gain affecting all target probes. Heterozygous loss affecting the digestion control probe at 132 nt and the reference probes at 238 and 398 nt.

Various related products

• SALSA MLPA Probemix ME042 CIMP: Contains 31 MS-MLPA targeting the promoter regions of the CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1 genes.
• SALSA MLPA Probemix P003 MLH1/MSH2: Lynch syndrome: Contains probes for MLH1 and MSH2.
• SALSA MLPA Probemix P008 PMS2: Lynch syndrome: Contains probes for PMS2.
• SALSA MLPA Probemix P043 APC: Contains probes for APC.
• SALSA MLPA Probemix P072 MSH6-MUTYH: Contains probes for MSH6, MUTYH and EPCAM.
• SALSA MLPA Probemix P248 MLH1-MSH2 Confirmation: Results obtained with P003 (MLH1 and MSH2) can be confirmed with this probemix.
• SALSA MLPA Probemix P378 MUTYH: Contains probes for MUTYH, SCG5 and GREM1.

General MLPA resources

• MS-MLPA technique overview
• Coffalyser.Net overview

MLPA education

• Getting started with MLPA
• Using Coffalyser.Net to analyse MLPA data
• Help centre with other learning resources

References

• Bouzourene H et al. (2010). Selection of patients with germline MLH1 mutated Lynch syndrome by determination of MLH1 methylation and BRAF mutation. Fam Cancer. 9:167-72.
• Cunningham JM et al. (1998). Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res. 58:3455-60.
• Deng G et al. (1999). Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res. 59:2029-33.
• Esteller M et al. (1998). MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas. Oncogene. 17:2413-7.
• Goel A et al. (2011). De novo constitutional MLH1 epimutations confer early-onset colorectal cancer in two new sporadic Lynch syndrome cases, with derivation of the epimutation on the paternal allele in one. Int J Cancer. 128:869-78.
• Herman JG et al. (1998). Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A. 95:6870-5.
• Kane MF et al. (1997). Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 57:808-11.
• Kuiper RP et al. (2011). Recurrence and variability of germline EPCAM deletions in Lynch syndrome. Hum Mutat. 32:407-14.
• Ligtenberg MJL et al. (2009). Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3' exons of TACSTD1. Nat Genet. 41:112-7.
• Morak M et al. (2018). Comprehensive analysis of the MLH1 promoter region in 480 patients with colorectal cancer and 1150 controls reveals new variants including one with a heritable constitutional MLH1 epimutation. J Med Genet. 55:240-8.
• Niessen RC et al. (2009). Germline hypermethylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome. Genes Chromosomes Cancer. 48:737-44.
• Pérez-Carbonell L et al. (2010). Methylation analysis of MLH1 improves the selection of patients for genetic testing in Lynch syndrome. J Mol Diagn. 12:498-504.
• Pinto D et al. (2018). Contribution of MLH1 constitutional methylation for Lynch syndrome diagnosis in patients with tumor MLH1 downregulation. Cancer Med. 7:433-44.
• Simpkins SB et al. (1999). MLH1 promoter methylation and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers. Hum Mol Genet. 8:661-6.