Intended use: This SALSA MLPA probemix P072 MSH6-MUTYH is an in vitro diagnostic (IVD)
1 or a research use only (RUO) assay for the detection of exon deletions or duplications in the
MSH6 gene and
EPCAM/MSH2 region in order to confirm a potential cause and clinical diagnosis for Lynch syndrome. In addition, this assay can detect deletions or duplications and the p.Y179C and p.G396D point mutations in the
MUTYH gene, in order to confirm a potential cause and clinical diagnosis for MUTYH-Associated Polyposis (MAP). The assay can also be used for molecular genetic testing of at-risk family members.
This assay is for use with human DNA extracted from peripheral blood. Deletions or duplications detected with the P072 MSH6-MUTYH probemix must be verified by another technique. In particular, deletions or duplications detected by only a single probe always require validation by another method. Most defects in the aforementioned 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. Of note, not all exons for
EPCAM,
MSH2 and
MUTYH are covered in this probemix. This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test are intended to be interpreted by a clinical molecular geneticist or equivalent.
This probemix can be used on tumour tissue in a research setting.
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: Lynch syndrome (LS) is characterised by an increased risk of colorectal cancer (CRC; life time risk 52-82%), endometrial cancer (life time risk 25-60%), ovarian cancer (life time risk 4-12%) and other cancers such as stomach and small intestine. It is caused by a heterozygous pathogenic variant in one of the mismatch repair (MMR) genes:
MLH1 and
MSH2 are responsible for ~90% of cases,
MSH6 for ~7-10% and
PMS2 for <5%. Germline deletions of the 3’ end of
EPCAM results in inactivation of
MSH2 by hypermethylation of the
MSH2 promoter, and is the cause of LS in ~1% of cases (Ligtenberg et al. 2009). 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).
The risk of CRC associated with
MLH1 and
MSH2 pathogenic variants is significantly higher and the mean age of onset for CRC is younger, compared to
MSH6 or
PMS2 pathogenic variants. Endometrial cancer is commonly observed in females with an
MSH6 pathogenic variant, and can be the first presented tumour.
It has been shown that 3’ end
EPCAM deletions result in comparable risks of CRC as
MSH2 mutations. However, the risk of developing endometrial cancer might depend on the extent of the deletion, where carriers with a deletion extending close to the
MSH2 promoter region have a higher risk than carriers with a deletion of only the 3’ end of
EPCAM (Kempers et al. 2011).
More information on Lynch syndrome is available on
https://www.ncbi.nlm.nih.gov/books/NBK1211/.
Biallelic pathogenic mutations of
MUTYH cause MUTYH-Associated Polyposis (MAP). MAP is characterised by an increased lifetime risk of CRC (43% to almost 100% in the absence of timely surveillance). Typically, MAP is associated with 10 to a few 100 colonic adenomatous polyps that are evident at a mean age of about 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. The two most common
MUTYH mutations associated with hereditary colorectal cancer are p.Y179C (c.536A>G) in exon 7 and p.G396D (c.1187G>A) in exon 13. These two mutations comprise approximately 80% of all germline alterations found in patients of Northern European origin. In Eastern Asian populations, these hotspot mutations are (almost) not prevalent (Aretz et al. 2013).
More information on MAP is available on:
https://www.ncbi.nlm.nih.gov/books/NBK107219/
Probemix content: This SALSA MLPA probemix P072 MSH6-MUTYH
contains 48 probes with amplifications products between 130 and 500 nt (Table 1 of the product description), including 12 reference probes. At least one MLPA probe is present for each exon of
MSH6. For
MUTYH all exons are covered by a probe, except exons 3, 7, 10, 12 and 14. Moreover, two mutation specific probes are present for
MUTYH p.Y179C and p.G396D, which will only generate a signal when the respective mutations are present. In addition, five probes detect the
EPCAM gene, including two exon 9 probes. Finally, three probes have been included targeting sequences in, down- and upstream of the
MSH2 gene. The identity of the genes detected by the reference probes is available online (
www.mlpa.com) and in Table 2c of the product description.
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 SD022: The SD022 Binning DNA provided with this probemix can be used as Binning DNA sample for binning of two mutation-specific probes in
MUTYH (184 nt, 18416-SP0654-L23441, detecting the p.Y179C (c.536A>G) mutation; and probe 258 nt, 21267-SP0655-L23442, detecting the p.G396D (c.1187G>A) mutation). SD022 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 SD022 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), as for this purpose true mutation/SNP 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 SD022 Binning DNA product description.
Sample DNA
Sample DNA developed for this product: