Intended purpose
The SALSA MLPA Probemix P003 MLH1/MSH2 is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in specific regions of the MLH1, MSH2 and EPCAM genes, as well as a recurrent 10 Mb inversion on chromosome arm 2p which disrupts the MSH2 gene, in genomic DNA isolated from human peripheral whole blood specimens. P003 MLH1/MSH2 is intended to confirm a potential cause for and clinical diagnosis of Lynch syndrome and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P003 MLH1/MSH2 should be confirmed with the SALSA MLPA Probemix P248 MLH1-MSH2 Confirmation assay or a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in the MLH1 and MSH2 genes are point mutations, none of which will be detected by MLPA. P248 MLH1-MSH2 Confirmation cannot be used to verify deletions or duplications in the EPCAM gene. It is therefore recommended to use this assay in combination with sequence analysis.

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, pre-implantation or prenatal testing, population screening, or 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 SD052 Binning DNA.

Clinical background
Lynch syndrome, 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, endometrial cancer, ovarian cancer, gastric cancer and small bowel 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: MLH1, MSH2, MSH6 or PMS2. Another cause of Lynch syndrome is a deletion of the 3’ part of EPCAM, leading to constitutional epigenetic silencing of the downstream MSH2 gene (Lynch et al. 2015). The estimated contribution of the different genes to Lynch syndrome is 15-40% for MLH1, 20-40% for MSH2, 12-35% for MSH6, 5-25% for PMS2 and <10% for EPCAM. More information about Lynch syndrome is available on http://www.ncbi.nlm.nih.gov/books/NBK1211/.

Among the various defects in the MLH1 and MSH2 genes that have been found in patients, deletions and duplications of complete exons are usually missed by standard sequence analysis. The MLPA technique can detect most of these deletions and duplications and therefore complements sequence analysis of the MLH1 and MSH2 genes.

Probemix content
The SALSA MLPA Probemix P003-D1 MLH1/MSH2 contains 50 MLPA probes with amplification products between 130 and 499 nucleotides (nt). This includes 19 probes for the MLH1 gene, 18 probes for the MSH2 gene and two probes for the EPCAM gene (formerly known as TACSTD1). EPCAM is a gene located just upstream of MSH2. Deletions of the last exon of EPCAM can result in silencing of the MSH2 gene. Furthermore, this probemix contains two probes specific for the recurrent 10 Mb inversion that disrupts the MSH2 gene (Wagner et al. 2002, Chen 2008, Rhees et al. 2014), which will only generate a signal when the inversion is present. In addition, nine reference probes are included that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available 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 at www.mrcholland.com.

SALSA Binning DNA SD052
The SD052 Binning DNA provided with this probemix can be used for binning of all probes including the two inversion-specific probes (265 nt probe 20091-SP0917-L28216 and 317 nt probe 20090-SP0916-L28222 for a 10 Mb inversion with one breakpoint in intron 7 of MSH2). SD052 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 SD052 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). 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 SD052 Binning DNA product description, available online: www.mrcholland.com.

Sample DNA
Sample DNA developed for this product:

• SD052 Binning DNA - included with this probemix.