Intended use: 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 EPCAM. 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: 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: The SALSA MLPA Probemix P003-D1 MLH1/MSH2 contains 50 MLPA probes with amplification products between 130 and 499 nucleotides (nt). This includes 39 probes for the MLH1/MSH2/EPCAM gene regions. Furthermore, this probemix also contains two probes specific for the recurrent 10 Mb inversion that disrupts MSH2 (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.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 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 synthetic DNA that contains the target sequence detected by the above mentioned probe. 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 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 provided.

Sample DNA
Sample DNA developed for this product:

• SD052 Binning DNA - included with this probemix.