The SALSA MLPA probemix P008 PMS2 is an in vitro diagnostic (IVD)1
or a research use only (RUO) assay for the detection of deletions or duplications in exons 1-11 of PMS2
and the detection of deletions or duplications in exons 12-15 of PMS2
in order to confirm a potential cause and clinical diagnosis of Lynch syndrome or constitutional MMR-deficiency syndrome. 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 not for use with DNA extracted from formalin-fixed paraffin embedded or fresh tumour materials.
Deletions or duplications detected with the P008 PMS2 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 PMS2
gene are point mutations, none of which will be detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of the PMS2
gene. Suitable reference samples should be identified for proper data analysis. To determine whether a deletion or duplication of exons 12-15 is present in PMS2 OR PMS2CL
, MLPA should always be combined with other methods, for example gene-specific long range PCR and sequencing of the amplification products. 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).
Heterozygous germ line mutations in the PMS2
gene (a mismatch repair gene) are a possible cause of Lynch syndrome (LS; also referred to as hereditary predisposition to non-polyposis colorectal cancer - HNPCC), an autosomal dominant disorder. LS is an inherited cancer of the digestive tract, particularly the colon and rectum. In addition, it increases the risk for numerous other cancers due to impaired DNA repair. More information is available on http://www.ncbi.nlm.nih.gov/books/NBK1211/
Among the various defects in the PMS2
gene 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 PMS2
Between 10-20% of LS cases are due to mutations in PMS2
. 80-90% of cases are due to mutations in MLH1
and 3% of cases are due to mutations in EPCAM
(Tiwari et al. 2016). See pages 10 & 11 for several publications on probemix P008 PMS2.
Absence or inactivation of both PMS2
alleles, resulting in paediatric intestinal cancer and polyposis (constitutional MMR-deficiency syndrome; CMMR-D), has been described but is extremely rare (Herkert et al. 2011).
This SALSA MLPA probemix P008 PMS2 contains 47 MLPA probes with amplification products between 128 and 483 nt: 34 probes for the PMS2/PMS2CL
genes (Table 2) and 13 reference probes that detect sequences outside this region. The identity of the genes detected by the reference probes is available online (www.mlpa.com
Detection of abnormal copy numbers of PMS2
exons is complicated due to the existence of many pseudogenes. In exons 12-15 no reliable sequence differences exist between PMS2
and the PMS2CL
pseudogene (van der Klift et al. 2010). As a consequence, it is not possible to design MLPA probes that are 100% specific for these PMS2
exons. However, the combined copy number of PMS2
is very consistent among healthy individuals. For this reason, five probes are included that detect sequences present in exons 12-15 of both PMS2
. As these probes detect both genes, a normal individual has 4 copies per cell and a deletion or duplication of one copy results in a probe ratio of 0.75 or 1.25, respectively. Together with the exon 1-11 probes, the analysis of these probes should exclude PMS2
copy number changes in the great majority of samples tested.
In case one or more of these five probes do indicate a copy number change in the exon 12-15 region, it should be determined whether the copy number change is in PMS2
or in PMS2CL
. To facilitate this, 10 probes are included that detect the copy number of both allelic forms of five SNPs in exons 11-15 of PMS2
. The distribution of these SNP alleles among PMS2
varies. More information can be found in the “Interpretation of results” section, and in the appendix. Analysis of these SNP-specific probes, complemented with gene-specific long range PCR and (next generation) sequencing analysis, can give further indications on whether a copy number change of exons 12-15 is in the PMS2
gene or in PMS2CL
(Li et al. 2015, Vaughn et al. 2011). As reported by Vaughn et al. (2011 and 2013), the great majority of deletions detected in exons 12-15 are in fact located in the PMS2
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 Reference Selection DNA SD072:
The selection of suitable reference DNA samples that can be used with P008 PMS2 is complicated. To facilitate the selection of suitable reference DNA samples from your own sample collection, a reference selection DNA sample (catalogue number SD072) can be ordered from MRC-Holland. Reference selection DNA SD072 should only be used for initial experiments on DNA samples from healthy individuals with the intention to identify suitable reference samples. SD072 should not be used in all experiments as a reference sample.
For further details, consult the Reference Selection DNA SD072 product description, available online: www.mlpa.com
. This product, when used in combination with P008, is for in vitro diagnostic (IVD) purposes, in the countries specified at the end of the P008 product description.
Sample DNA developed for this product: