Intended purpose
The SALSA MLPA Probemix P043 APC is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² 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.

Copy number variations (CNVs) detected with P043 APC should be confirmed with a different technique. In particular, CNVs detected by only a single probe as well as the two MUTYH point mutations always require confirmation by another method. Most defects in the APC and MUTYH genes are point mutations, which will not be detected by MLPA, with exception of the two aforementioned MUTYH point mutations. It is therefore recommended to use this assay in combination with sequence analysis. FAP has a high incidence of mosaicism and mosaic APC mutations may not be detectable in blood.

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 SALSA Binning DNA SD022.

Clinical background
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); see Table 2 for more details). 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 (see Table 2 for more details).

Probemix content
The SALSA MLPA Probemix P043-E1 APC contains 45 MLPA probes with amplification products between 130 and 483 nt. This includes 29 probes for the APC gene, four probes for the MUTYH gene, and two probes for the region upstream of GREM1. This probemix also contains two probes specific for the c.536A>G (p.Tyr179Cys) and c.1187G>A (p.Gly396Asp) MUTYH mutations, which will only generate a signal when the mutation is present. In addition, eight 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.mrcholland.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.mrcholland.com.

SALSA Binning DNA SD022
The SALSA Binning DNA SD022 provided with this probemix can be used for binning of all probes, including the two MUTYH mutation-specific probes: the 188 nt probe 18416-SP0654-L29811, detecting the c.536A>G (p.Tyr179Cys) mutation; and the 193 nt probe 21267-SP0655-L23442, detecting the c.1187G>A (p.Gly396Asp) mutation. The 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 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). 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 SALSA Binning DNA SD022 product description, available online: www.mrcholland.com.


Sample DNA
Sample DNA developed for this product:

• SD022 Binning DNA - included with this probemix.