Intended use: This SALSA MLPA probemix P003 MLH1/MSH2 is an in vitro diagnostic (IVD)¹ or research use only (RUO) assay for the detection of exon deletion(s) or duplication(s) 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 order to confirm a potential cause and clinical diagnosis for Lynch syndrome. This product can also be used for molecular genetic testing of at-risk family members.

This assay is for use with peripheral blood derived genomic DNA and not for use with DNA extracted from formalin-fixed paraffin embedded or fresh tumour materials. Deletions or duplications detected with the P003 MLH1/MSH2 probemix should be verified by using the SALSA MLPA probemix P248 MLH1/MSH2 Confirmation or a different technique. This verification must always be performed for copy number changes detected by only a single probe. P248 MLH1/MSH2 Confirmation cannot be used to verify deletions or duplications in EPCAM.

This assay is not intended to be used as a standalone assay for clinical decisions. Most defects in the MLH1 and MSH2 genes are point mutations, the majority of which will not be detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis. 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 use (IVD) in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO).

Clinical background: Germline defects in the MLH1 and MSH2 genes are the most frequent cause of a hereditary predisposition to Lynch syndrome (formerly known as hereditary non-polyposis colorectal cancer; HNPCC). Lynch syndrome is an inherited cancer of the digestive tract, particularly the colon and rectum. In addition, defects in the MLH1 and MSH2 genes increase the risk for numerous other cancers due to impaired DNA repair. More information is available on http://www.ncbi.nlm.nih.gov/books/NBK1211/.

Approximately 50% of Lynch syndrome cases are attributed to mutations in MLH1, 40% are attributed to mutations in MSH2, and 1-3% are due to EPCAM deletions. In addition, 10-20% of the cases can be explained by mutations in the MSH6 and PMS2 genes. Among the various defects in the MLH1 and MSH2 genes that have been found in patients are deletions and duplications of complete exons, which 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: This SALSA MLPA probemix P003 MLH1/MSH2 version D1 contains 50 MLPA probes with amplification products between 130 and 499 nt (Table 1) including 39 probes for the MLH1/MSH2/EPCAM gene regions (Table 2), 2 probes that only generate signals on samples containing the recurrent 10 Mb inversion that disrupts MSH2 (Wagner et al. 2002, Chen 2008, Rhees et al. 2014), and 9 reference probes that detect sequences outside this region. The identity of the genes detected by the reference probes is available online (www.mlpa.com).

One probe is present for each of the 19 exons of the MLH1 gene and for each of the 16 exons of the MSH2 gene. Two probes are included for exon 9 of EPCAM (formerly known as TACSTD1), a gene located just upstream of MSH2. Deletions of this last exon of EPCAM can result in silencing of the MSH2 gene.

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.mlpa.com.

SALSA Binning DNA SD052: The SD052 Binning DNA provided with this probemix can be used as Binning DNA sample for binning of two inversion specific probes (265 nt probe 20091-SP0917-L28216 and 317 nt probe 20090-SP0916-L28222 for a 10 Mb inversion with breakpoint in intron 7 of MSH2). SD052 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequences 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 signals, as for this purpose true inversion positive patient samples or cell lines should be used. It is strongly advised to use DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For further details, please consult the SD052 Binning DNA product description provided.

Sample DNA
Sample DNA developed for this product:

• SD052 Binning DNA - included with this probemix.