The SALSA MLPA Probemix P043 APC is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative assay for the detection of deletions or duplications in the APC and MUTYH genes, and duplications in the upstream region of the GREM1 gene in order to confirm a potential cause for and clinical diagnosis of 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 people from European descent, c.536A>G (p.Tyr179Cys) and c.1187G>A (p.Gly396Asp), can be detected with this probemix. P043 APC can also be used for molecular genetic testing of at-risk family members. This assay is for use with genomic DNA extracted from human peripheral whole blood specimens.

For the full intended purpose, see the product description.

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, acquired 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 (CRC) 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 at http://www.ncbi.nlm.nih.gov/books/NBK1345/.

Mutations in the MUTYH gene also result in a hereditary predisposition to colon and gastric cancer, which is referred to as MAP. In contrast to the APC-associated disease FAP, MAP is an autosomal recessive trait and is considered less severe: polyps do not appear until adulthood and are less numerous than those found in patients with APC gene mutations. Nevertheless, phenotypes of APC- and MUTYH-related CRC partly overlap. Therefore, six probes for the MUTYH gene are included in this P043-E1 APC probemix, two of which will only generate a signal when the common c.536A>G (p.Tyr179Cys) or c.1187G>A (p.Gly396Asp) mutations are present. The c.536A>G (p.Tyr179Cys) and c.1187G>A (p.Gly396Asp) variants are carried by ~1%-2% of the general population and account for ≥90% of all MUTYH pathogenic variants in northern European populations. Up to 70% of MAP patients harbours at least one of these variants (Aretz et al. 2013). Since the MUTYH gene is small (11 kilobases (kb)), the four copy number detection MLPA probes are expected to detect a substantial part of MUTYH copy number changes. For instance, the most frequent CNV in MUTYH – a deletion of exon 4-16 that is reported in multiple patients (Castillejo et al. 2014) – can be detected with two probes in this probemix. More information on MAP is available at http://www.ncbi.nlm.nih.gov/books/NBK107219/. For complete exon coverage of MUTYH the SALSA MLPA Probemix P378 MUTYH is available.

A recurrent duplication of ~40 kb directly upstream of the GREM1 gene is known to lead to HMPS1. Patients with HMPS1 have a predisposition for developing CRC (Jaeger et al. 2012). Presence of this duplication is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that underlies tumorigenesis in juvenile polyposis of the large bowel. Several additional duplications in the GREM1 upstream region have been found: e.g. a duplication of the upstream region and the whole GREM1 gene of ~57 kb has been described in one patient with sigmoid colon carcinoma (Venkatachalam et al. 2011); a duplication of ~16 kb has been described in members of a family presenting with atypical FAP (Rohlin et al. 2016); and a duplication of ~24 kb in a patient with multiple colon polyps has been reported (McKenna et al. 2019). Two probes for the relevant region directly upstream of GREM1 are included in this probemix; both probes detect the 40 kb, 57 kb and 24 kb duplications and the 16 kb duplication can be detected by one of these probes (probe number 21272-L23310 at 217 nucleotides (nt)). For extended coverage of the GREM1 region the SALSA MLPA Probemix P378 MUTYH is available. 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. It is expected that 8-12% of all APC mutations in most populations are large rearrangements that can be detected with this MLPA probemix (Jarry et al. 2011; Kerr et al. 2013). Of note, several clinically relevant rearrangements of only the APC promoter region are reported (Snow et al. 2014; Kadiyska et al. 2014; Kalbfleisch et al. 2015; Marabelli et al. 2017), all of which can be detected with this probemix.

SALSA MLPA Probemix P043 APC is CE-marked under the IVDD for in vitro diagnostic (IVD) use in Europe. This assay has also been registered for IVD use in Costa Rica and Israel.

This assay is for research use only (RUO) in all other territories.

SALSA Binning DNA SD022 is an artificial DNA sample with a signal for all probes in the P043 APC probemix. Inclusion of a reaction with SD022 in initial experiments and in experiments following a change in electrophoresis conditions is recommended to aid in the creation of a bin set that links peaks to the probes that produce them. Binning DNA cannot be used as a reference sample in the MLPA data analysis, and cannot be used to quantify the signals of mutation-specific probes.

A vial of SALSA Binning DNA SD022 is included with every order of the P043 APC probemix, but it is possible to order additional vials separately.

For more information, see the product description.

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below have been tested with the current (E1) version of this product and have been shown to produce useful results.

APC

• Coriell NA14234: Heterozygous deletion affecting all APC probes.

MUTYH

• Coriell HG00097, Coriell HG01095, Coriell HG01519, Coriell HG01685, Coriell HG02224, Coriell NA19789, Coriell NA20522, Coriell NA20759: Positive for the MUTYH c.1187G>A (p.Gly396Asp) mutation.
• Coriell HG01918: Positive for the MUTYH c.536A>G (p.Tyr179Cys) mutation.

GREM1

• Coriell NA03184: Heterozygous duplication affecting both GREM1-up probes.

Products for Lynch syndrome and polyposis syndrome from MRC Holland

MRC Holland offers various different assays for Lynch syndrome and polyposis syndrome. The table below indicates which product can be used for which target gene(s).

Related digitalMLPA probemixes

Target many more regions simultaneously using our digitalMLPA technology, which combines the trusted MLPA technology with the power of NGS.

• D001 Hereditary Cancer Panel 1: Targets 28 genes involved in hereditary predisposition to cancer, including MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH.

Key related products

• P072 MSH6-MUTYH: Targets MSH6 and EPCAM/MSH2, involved in Lynch syndrome, and MUTYH, involved in MUTYH-associated polyposis (MAP).
• P378 MUTYH: Targets MUTYH, involved in MUTYH-associated polyposis (MAP), and the SCG5/GREM1 region, involved in hereditary mixed polyposis syndrome type 1 (HMPS1).
• P003 MLH1/MSH2: Targets MLH1, MSH2, and EPCAM, involved in Lynch syndrome. Intended for primary screening.
• P248 MLH1-MSH2 Confirmation: Targets MLH1 and MSH2, involved in Lynch syndrome. Can be used to confirm P003 MLH1/MSH2 results.
• P008 PMS2: Targets PMS2, involved in Lynch syndrome.
• ME011 Mismatch Repair Genes: Methylation status of the promoter regions of MLH1, MSH2, MSH6, and PMS2. Also targets EPCAM copy number and the BRAF p.V600E mutation.
• ME042 CIMP: Methylation status of the promoter regions of CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1.

General MLPA resources

• MLPA technique overview
• Coffalyser.Net overview

MLPA education

• Getting started with MLPA
• Using Coffalyser.Net to analyse MLPA data
• Help centre with other learning resources

Selected publications using P043 APC

• Araujo LF et al. (2019). Molecular basis of familial adenomatous polyposis in the southeast of Brazil: identification of six novel mutations. Int J Biol Markers. 34:80-9.
• Fujita M et al. (2022). Population-based Screening for Hereditary Colorectal Cancer Variants in Japan. Clin Gastroenterol Hepatol. 20:2132-41.
• Khan N et al. (2017). Novel mutations and phenotypic associations identified through APC, MUTYH, NTHL1, POLD1, POLE gene analysis in Indian Familial Adenomatous Polyposis cohort. Sci Rep. 7:2214.
• Nguyen BH et al. (2022). The Mutation Spectrum and Two Novel Point Mutations in the APC Gene in Vietnamese Patients with Familial Adenomatous Polyposis. Asian Pac J Cancer Prev. 23:1517-22.
• Snow AK et al. (2015). APC promoter 1B deletion in seven American families with familial adenomatous polyposis. Clin Genet. 88:360-5.
• Tanaka K et al. (2022). Small Intestinal Involvement and Genotype-Phenotype Correlation in Familial Adenomatous Polyposis. Tech Innov Gastrointest Endosc. 24:26-34.
• Wang D et al. (2019). Adenomatous Polyposis Coli Gene Mutations in 22 Chinese Pedigrees with Familial Adenomatous Polyposis. Med Sci Monit. 25:3796-803.

References

• Aretz S et al. (2013). Clinical utility gene card for: MUTYH-associated polyposis (MAP), autosomal recessive colorectal adenomatous polyposis, multiple colorectal adenomas, multiple adenomatous polyps (MAP) - update 2012. Eur J Hum Genet. 21.
• Castillejo A et al. (2014). Prevalence of germline MUTYH mutations among Lynch-like syndrome patients. Eur J Cancer. 50:2241-50.
• Jaeger E et al. (2012). Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1. Nat Genet. 44:699-703.
• Jarry J et al. (2011). A survey of APC mutations in Quebec. Fam Cancer. 10:659-65.
• Kadiyska TK et al. (2014). APC promoter 1B deletion in familial polyposis--implications for mutation-negative families. Clin Genet. 85:452-7.
• Kalbfleisch T et al. (2015). Characterization of an APC Promoter 1B deletion in a Patient Diagnosed with Familial Adenomatous Polyposis via Whole Genome Shotgun Sequencing. F1000Res. 4:170.
• Kerr SE et al. (2013). APC germline mutations in individuals being evaluated for familial adenomatous polyposis: a review of the Mayo Clinic experience with 1591 consecutive tests. J Mol Diagn. 15:31-43.
• Marabelli M et al. (2017). A novel APC promoter 1B deletion shows a founder effect in Italian patients with classical familial adenomatous polyposis phenotype. Genes Chromosomes Cancer. 56:846-54.
• McKenna DB et al. (2019). Identification of a novel GREM1 duplication in a patient with multiple colon polyps. Fam Cancer. 18:63-6.
• Rohlin A et al. (2016). GREM1 and POLE variants in hereditary colorectal cancer syndromes. Genes Chromosomes Cancer. 55:95-106.
• Snow AK et al. (2015). APC promoter 1B deletion in seven American families with familial adenomatous polyposis. Clin Genet. 88:360-5.
• Venkatachalam R et al. (2011). Identification of candidate predisposing copy number variants in familial and early-onset colorectal cancer patients. Int J Cancer. 129:1635-42.