The SALSA MLPA probemix P043 APC is an in vitro diagnostic (IVD)1
or a research use only (RUO) assay for the detection of deletions or duplications in the human APC
gene, and the upstream region of the GREM1
gene, in order to confirm a potential cause and clinical diagnosis for familial adenomatous polyposis (FAP), MUTYH
-associated polyposis (MAP), or hereditary mixed polyposis syndrome (HMPS1), respectively. In addition, the presence of the two most common point mutations in the MUTYH
gene among Europeans can be detected by the P043 probemix. This product 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 and is not for use with DNA extracted from formalin-fixed paraffin embedded materials or fresh tumour material. Deletions or duplications detected with the P043 APC probemix should 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 APC
gene are point mutations, the majority of which will not detected by MLPA, with exception of the two most common MUTYH
mutations among Europeans. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of these genes. In addition, there is a high incidence of mosaicism in FAP patients and mosaic APC
mutations may not be detectable in blood or other healthy tissues. This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test must be interpreted by a clinical molecular geneticist or equivalent.
Please 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).
Germline defects in the APC
gene are the most frequent cause of a hereditary predisposition to polyposis colon cancer, which can present as familial adenomatous polyposis (FAP), attenuated FAP (AFAP), or gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS). In addition, mutations in the APC
gene are an initiating event for sporadic colorectal tumour development. Most FAP patients develop adenomatous colonic polyps in the first two decades of life and in untreated individuals this progresses into colonic cancer at an average age of 39 years. APC
-related colorectal cancer is a dominant
trait. Approximately 4% of (A)FAP patients have a somatic mosaic APC
mutation, which increases to ~20% when only de novo
cases are considered. More information on APC
-associated polyposis is available on http://www.ncbi.nlm.nih.gov/books/NBK1345/
Mutations in the MUTYH
gene result in a hereditary predisposition to colon and gastric cancer. In contrast to the APC
-associated colorectal cancer is an autosomal recessive
trait. Polyps caused by mutations in the MUTYH
gene do not appear until adulthood and are less numerous than those found in patients with APC
gene mutations. As the phenotypes of APC
- and MUTYH
-related colorectal cancer overlap, six probes for the MUTYH
gene are included in this P043-E1 probemix, two of which will only generate a signal when the common c.536A>G (p.Tyr179Cys) or c.1187G>A (p.Gly396Asp) mutation is present; these two mutations account for ~80% of all MUTYH
pathogenic variants among Europeans. Since the MUTYH
gene is very small (11 kb), the four wildtype-specific probes are expected to detect a substantial part of MUTYH
copy number changes. More information on MUTYH
-associated polyposis is available on http://www.ncbi.nlm.nih.gov/books/NBK107219/
. For complete exon coverage of MUTYH
probemix P378 is available.
A recurrent duplication of 40 kb close to the GREM1
gene is known to be linked to an increased risk of developing colorectal cancer. This syndrome is known as hereditary mixed polyposis syndrome (HMPS1). Presence of this duplication is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel (Jaeger at al. 2012). Another duplication of ~16 kb in this region has been described more recently in members of a family presenting with atypical FAP (Rohlin et al. 2015). Furthermore, a duplication of the complete GREM1
gene, including the upstream region (~57 kb in total), has been described in one patient with sigmoid colon carcinoma (Venkatachalam et al. 2011). Two probes are included in the P043 probemix to detect the 40 kb and 57 kb duplications. The 16 kb duplication can be detected by one of these probes. More information on HMPS1 is available at http://omim.org/entry/601228.
Among the various defects in the APC
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 APC
genes. It is expected that 6-12% of all APC
mutations in most populations are large rearrangements that can be detected with this MLPA probemix (Kerr et al. 2013; Jarry et al. 2011). See ‘Selected publications’ below for relevant scientific articles on probemix P043 APC.
This SALSA MLPA probemix P043 APC contains 45 MLPA probes with amplification products between 130 and 483 nt: 29 probes for the APC
gene, 6 probes for the MUTYH
gene, 2 GREM1
upstream probes and 8 reference probes detecting autosomal chromosomes. The identity of the genes detected by the reference probes is available online (www.mlpa.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.mlpa.com
SALSA Binning DNA SD022:
The SD022 Binning DNA provided with this probemix can be used for binning of the two mutation-specific probes in the MUTYH
gene (probe 18416-SP0654-L29811, detecting the c.536A>G (p.Tyr179Cys) mutation; and probe 21267-SP0655-L23442, detecting the c.1187G>A (p.Gly396Asp) 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 probes. 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 positive reference in the MLPA data analysis, neither should it be used in quantification of mutation signals. For further details, please consult the SD022 Binning DNA product description, available online: www.mlpa.com