SALSA MLPA P043 APC probemixCE

application: Hereditary Polyposis Colon Cancer
region: APC, 5q21-q22
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version: E1
sold since: 2017-03-13

item no. description price
P043-025R SALSA MLPA P043 APC probemix – 25 rxn € 237
P043-050R SALSA MLPA P043 APC probemix – 50 rxn € 474
P043-100R SALSA MLPA P043 APC probemix – 100 rxn € 948
EK1-FAM SALSA MLPA EK1 reagent kit – 100 rxn - FAM € 294
EK1-Cy5 SALSA MLPA EK1 reagent kit – 100 rxn - Cy5 € 294
EK5-FAM SALSA MLPA EK5 reagent kit – 500 rxn - FAM € 1355
EK5-Cy5 SALSA MLPA EK5 reagent kit – 500 rxn - Cy5 € 1355

Please note that both a probemix and reagent kit are needed to perform MLPA.

description
Intended use: This 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, MUTYH 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 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 optimised for use with human DNA derived from peripheral blood and is not for use with DNA extracted from formalin-fixed paraffin embedded or fresh tumour materials.

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 and MUTYH gene 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 these genes. 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 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 APC gene are the most frequent cause of a hereditary predisposition to polyposis colon cancer. In addition, mutations in the APC gene are an initiating event for sporadic colorectal tumour development. APC-related colorectal cancer is a dominant trait. 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 gene, MUTYH-associated colorectal cancer should be regarded as 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. More information on MUTYH-associated polyposis is available on http://www.ncbi.nlm.nih.gov/books/NBK107219/.

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 these will only generate a signal when the c.536A>G (p.Tyr179Cys) or c.1187G>A (p.Gly396Asp) mutation is present. 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.

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 tumourigenesis 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 and MUTYH 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 and MUTYH genes. The expected number of APC chromosomal rearrangements that can be detected with this MLPA probemix is around 6% of all APC mutations in most populations (Kerr et al. 2013; Jarry et al. 2011). See below for several publications on probemix P043 APC.

P043-E1 probemix content: 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. 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), three DNA Denaturation Fragments (D-fragments), and one chromosome X and one chromosome Y-specific fragment (Table 1). The Q-fragments are only visible when less than 100 ng sample DNA is used. Low signal of the 88 or 96 nt fragment indicates incomplete DNA denaturation. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol.

Binning DNA sample (included): SALSA Binning DNA SD022-S01 is included with each shipment of the P043 APC probemix and 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). Inclusion of one reaction with 5 µl SALSA Binning DNA SD022 in the initial MLPA experiment 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 conditions or experimental set-up have been changed (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 mutation positive patient samples or cell lines should be used. For further details, please consult the SALSA Binning DNA SD022 product description provided (also available online: www.mlpa.com).

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references
Pedace L, Majore S, Megiorni F, Binni F, De Bernardo C, Antigoni I, Preziosi N, Mazzilli MC, Grammatico P. (2008). Identification of a novel duplication in the APC gene using multiple ligation probe amplification in a patient with familial adenomatous polyposis.
Pagenstecher C, Gadzicki D, Stienen D, Uhlhaas S, Mangold E, Rahner N, Arslan-Kirchner M, Propping P, Friedl W, Aretz S. (2007). A Complex Rearrangement in the APC Gene Uncovered by Multiplex Ligation-Dependent Probe Amplification.
Cao X, Hong Y, Eu KW, Loi C, Cheah PY. (2006). Singapore Familial Adenomatous Polyposis (FAP) Patients with Classical Adenomatous Polyposis but Undetectable APC Mutations Have Accelerated Cancer Progression.
McCart A, Latchford A, Volikos E, Rowan A, Tomlinson I, Silver A. (2006). A Novel Exon Duplication Event Leading to a Truncating Germ-line Mutation of the APC Gene in a Familial Adenomatous Polyposis Family.
Renkonen ET, Nieminen P, Abdel-Rahman WM, Moisio AL, Jarvela I, Arte S, Jarvinen HJ, Peltomaki P. (2005). Adenomatous Polyposis Families That Screen APC Mutation-Negative by Conventional Methods Are Genetically Heterogeneous.
Mihalatos M, Apessos A, Dauwerse H, Velissariou V, Psychias A, Koliopanos A, Petropoulos K, Triantafillidis JK, Danielidis I, Fountzilas G, Agnantis NJ, Nasioulas G. (2005). Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients.
Michils G, Tejpar S, Thoelen R, van Cutsem E, Vermeesch JR, Fryns JP, Legius E, Matthijs G. (2005). Large deletions of the APC gene in 15% of mutation-negative patients with classical polyposis (FAP): a Belgian study.
Meuller J, Kanter-Smoler G, Nygren AO, Errami A, Gronberg H, Holmberg E, Bjork J, Wahlstrom J, Nordling M. (2004). Identification of genomic deletions of the APC gene in familial adenomatous polyposis by two independent quantitative techniques.
Bunyan DJ, Eccles DM, Sillibourne J, Wilkins E, Thomas NS, Shea-Simonds J, Duncan PJ, Curtis CE, Robinson DO, Harvey JF, Cross NC. (2004). Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification.

product history
version E1: Four new MUTYH probes and two probes for the GREM1 duplication region were added; one MUTYH probe and three reference probes have been removed.
version D1: Four extra APC probes have been included for the very long last exon and six refeence probes have been replaced.
version C1: Four new APC and three new MUTYH probes have been included. Five APC probes have been removed. In addition, the 88 and 96 nt control fragments have been replaced (QDX2).
version B1: Four APC probes have been removed and seven new APC probes have been included. In addition, four control fragments at 88-96-100-105 nt have been added

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