Intended purpose
The SALSA MLPA Probemix P072 MSH6-MUTYH is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² 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.

Copy number variations (CNVs) detected with P072 MSH6-MUTYH should be confirmed with a different technique. In particular, CNVs detected by only a single probe as well as the two MUTYH point mutations always require confirmation by another method. Most defects in the MSH6 gene, EPCAM/MSH2 region and the MUTYH gene are point mutations, which will not be detected by MLPA, with exception of the two aforementioned MUTYH point mutations. It is therefore recommended to use this assay in combination with sequence analysis. Of note, not all exons of EPCAM, MSH2 and MUTYH are covered in this probemix.

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, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, population screening, pre-implantation or prenatal testing. Only in a research setting can this device be used for the detection of, or screening for, acquired or somatic genetic aberrations, e.g. from DNA extracted from formalin-fixed paraffin embedded (FFPE) or fresh tumour materials.

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, and SALSA Binning DNA SD022.

Clinical background
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.

Probemix content
The SALSA MLPA Probemix P072-D1 MSH6-MUTYH contains 48 MLPA probes with amplification products between 130 and 500 nt. This includes 15 probes for the MSH6 gene, eleven probes for the MUTYH gene, five probes for the EPCAM gene and three probes that target sequences in, down- and upstream of the MSH2 gene. Furthermore, this probemix also contains two probes specific for the c.536A>G (p.Tyr179Cys) and c.1187G>A (p.Gly396Asp) MUTYH mutations, which will only generate a signal when the mutation is present. In addition, twelve reference probes are included that target relatively copy number stable regions in various cancer types associated with LS and MAP. Complete probe sequences and the identity of the genes detected by the reference probes are available in Table 3 and online (www.mrcholland.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 MLPA General Protocol and online (www.mrcholland.com).

SALSA Binning DNA SD022
The SALSA Binning DNA SD022 provided with this probemix can be used for binning of all probes, including the two MUTYH mutation-specific probes: the 184 nt probe 18416-SP0654-L23441, detecting the c.536A>G (p.Tyr179Cys) mutation; and the 258 nt probe 18417-SP0655-L23442, detecting the c.1187G>A (p.Gly396Asp) mutation. This 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 SD022 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). 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 SALSA Binning DNA SD022 product description, available online: www.mrcholland.com.

Sample DNA
Sample DNA developed for this product:

• SD022 Binning DNA - included with this probemix.