The SALSA MLPA Probemix P072 MSH6-MUTYH is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative assay for the detection of deletions or duplications in the MSH6 gene and the EPCAM/MSH2 region in order to confirm a potential cause of and clinical diagnosis for Lynch Syndrome (LS), as well as deletions and duplications in the MUTYH gene in order to confirm a potential cause of and clinical diagnosis for MUTYH-Associated Polyposis (MAP). In addition, the presence of the two most common point mutations in the MUTYH gene among people from European descent, c.536A>G (p.Tyr179Cys) and c.1187G>A (p.Gly396Asp), can be detected with this probemix. P072 MSH6-MUTYH is also intended for molecular genetic testing of at-risk family members. This assay is for use with genomic DNA isolated from human peripheral whole blood specimens.

For the full intended purpose, see the product description.

Lynch syndrome (LS; formerly known as hereditary non-polyposis colorectal cancer (HNPCC)), is an adult-onset hereditary cancer susceptibility syndrome predisposing to several cancer types, the most prevalent being colorectal cancer (CRC), endometrial cancer, gastric cancer and ovarian cancer. It is an autosomal dominantly inherited syndrome that is caused by heterozygous germline mutations in one of the four major DNA mismatch repair genes, i.e. MLH1, MSH2, MSH6 or PMS2. The estimated contribution of the different genes to LS is 15-40% for MLH1, 20-40% for MSH2, 12-35% for MSH6, and 5-25% for PMS2. Germline deletions of the 3’-end of EPCAM result in inactivation of MSH2 by hypermethylation of the MSH2 promoter, and are the cause of LS in <10% of cases (Ligtenberg et al. 2009; Lynch et al. 2015; Tiwari et al. 2016). Although it is expected that deletion of exon 9 of EPCAM (including the transcription stop site) would be sufficient to lead to hypermethylation of the MSH2 promoter, studies have shown that the identified EPCAM deletions included at least exon 8 and 9 (Kuiper et al. 2011, Rumilla et al. 2011). More information on Lynch syndrome is available on https://www.ncbi.nlm.nih.gov/books/NBK1211/.

Mutations in the MUTYH gene also result in a hereditary predisposition to colon and gastric cancer, which is referred to as MAP. In contrast to LS, MAP is an autosomal recessive disorder. In MAP patients, ten to several hundred colonic adenomatous polyps develop and these become evident at a mean age of 50 years. However, colon cancer can also develop in the absence of polyposis. A single defective copy of the MUTYH gene may result in no, or only a small increase in risk for CRC. Phenotypes of Lynch (like) syndrome and MAP can partly overlap. There are two common MUTYH mutations, c.536A>G (p.Tyr179Cys) and c.1187G>A (p.Gly396Asp), that are carried by ~1%-2% of the general population and account for ≥90% of all MUTYH pathogenic variants in northern European populations. Up to 70% of MAP patients harbor at least one of these variants (Aretz et al. 2013). Since the MUTYH gene is small (11 kilobases (kb)), the eleven copy number detection MLPA probes present in this P072 MSH6-MUTYH probemix are expected to detect most of the MUTYH copy number changes as 11/16 exons are covered. For instance, the most frequent CNV in MUTYH – a deletion of exon 4-16 that is reported in multiple patients (Castillejo et al. 2014) – can be detected with nine probes in this probemix. More information on MAP is available at http://www.ncbi.nlm.nih.gov/books/NBK107219/. For complete exon coverage of MUTYH the SALSA MLPA probemix P378 MUTYH is available.

SALSA MLPA Probemix P072 MSH6-MUTYH is CE-marked under the IVDD for in vitro diagnostic (IVD) use in Europe. This assay has also been registered for IVD use in Israel.

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

SALSA Binning DNA SD022 is an artificial DNA sample with a signal for all probes in the P072 MSH6-MUTYH probemix. Inclusion of a reaction with SD022 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 SD022 is included with every order of the P072 MSH6-MUTYH 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.

2p

• Coriell NA10401: Heterozygous duplication affecting the probes for EPCAM, MSH2, KCNK12 and MSH6, and two reference probes at 155 nt and 190 nt.
• Coriell NA13451: Heterozygous deletion affecting the probes for EPCAM, MSH2, KCNK12 and MSH6.

MUTYH

• Coriell HG00097, Coriell HG01095, Coriell HG01519: Positive for the MUTYH c.1187G>A (p.Gly396Asp) mutation.
• Coriell HG01918: Positive for the MUTYH c.536A>G (p.Tyr179Cys) mutation.

Products for Lynch syndrome and polyposis syndrome from MRC Holland

MRC Holland offers various different assays for Lynch syndrome and polyposis syndrome. The table below indicates which product can be used for which target gene(s).

Related digitalMLPA probemixes

Target many more regions simultaneously using our digitalMLPA technology, which combines the trusted MLPA technology with the power of NGS.

• D001 Hereditary Cancer Panel 1: Targets 28 genes involved in hereditary predisposition to cancer, including MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH.

Key related products

• P003 MLH1/MSH2: Targets MLH1, MSH2, and EPCAM, involved in Lynch syndrome. Intended for primary screening.
• P248 MLH1-MSH2 Confirmation: Targets MLH1 and MSH2, involved in Lynch syndrome. Can be used to confirm P003 MLH1/MSH2 results.
• P378 MUTYH: Targets MUTYH, involved in MUTYH-associated polyposis (MAP), and the SCG5/GREM1 region, involved in hereditary mixed polyposis syndrome type 1 (HMPS1).
• ME011 Mismatch Repair Genes: Methylation status of the promoter regions of MLH1, MSH2, MSH6, and PMS2. Also targets EPCAM copy number and the BRAF p.V600E mutation.
• P043 APC: Targets APC, involved in familial adenmatous polyposis (FAP), MUTYH, involved in MUTYH-associated polyposis, and the GREM1 region, involved in hereditary mixed polyposis syndrome (HMPS1).
• P008 PMS2: Targets PMS2, involved in Lynch syndrome.
• ME042 CIMP: Methylation status of the promoter regions of CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1.

General MLPA resources

• 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

Selected publications using P072 MSH6-MUTYH

• Carneiro da Silva F et al. (2015). Clinical and Molecular Characterization of Brazilian Patients Suspected to Have Lynch Syndrome. PLoS One. 10:e0139753.
• Dymerska D et al. (2017). New EPCAM founder deletion in Polish population. Clin Genet. 92:649-53.
• Kóder G et al. (2020). Identification of Novel Pathogenic Sequence Variants of the Mismatch Repair Genes During Screening for Lynch Syndrome in a Single Centre of Eastern Hungary. J Gastrointest Cancer. 51:1007-15.
• Marabelli M et al. (2020). Is tumor testing efficiency for Lynch syndrome different in rectal and colon cancer? Dig Liver Dis. 52:1503-11.
• Schneider NB et al. (2018). Germline MLH1, MSH2 and MSH6 variants in Brazilian patients with colorectal cancer and clinical features suggestive of Lynch Syndrome. Cancer Med. 7:2078-88.
• Soares BL et al. (2018). Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing. Fam Cancer. 17:387-94.
• Tsaousis GN et al. (2019). Analysis of hereditary cancer syndromes by using a panel of genes: novel and multiple pathogenic mutations. BMC Cancer. 19:535.
• Ziada-Bouchaar H et al. (2017). First description of mutational analysis of MLH1, MSH2 and MSH6 in Algerian families with suspected Lynch syndrome. Fam Cancer. 16:57-66.

References

• Aretz S et al. (2013). Clinical utility gene card for: MUTYH-associated polyposis (MAP), autosomal recessive colorectal adenomatous polyposis, multiple colorectal adenomas, multiple adenomatous polyps (MAP) - update 2012. Eur J Hum Genet. 21.
• Castillejo A et al. (2014). Prevalence of germline MUTYH mutations among Lynch-like syndrome patients. Eur J Cancer. 50:2241-50.
• 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.
• Lynch HT et al. (2015). Milestones of Lynch syndrome: 1895-2015. Nat Rev Cancer. 15:181-94.
• Rumilla K et al. (2011). Frequency of deletions of EPCAM (TACSTD1) in MSH2-associated Lynch syndrome cases. J Mol Diagn. 13:93-9.
• Tiwari AK et al. (2016). Lynch syndrome in the 21st century: clinical perspectives. QJM. 109:151-8.