Intended purpose:
The SALSA MLPA probemix P008 PMS2 is an in vitro diagnostic (IVD)¹ or a research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in exons 1-11 of the PMS2 gene and in exons 12-15 of the PMS2 or PMS2CL genes in genomic DNA isolated from human peripheral whole blood specimens. P008 PMS2 is intended to confirm a potential cause for and clinical diagnosis of Lynch syndrome or constitutional mismatch repair deficiency syndrome and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P008 PMS2 should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation 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 assay in combination with sequence analysis of the PMS2 gene. Suitable reference samples should be identified for proper data analysis. To determine whether a CNV 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.

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 Reference Selection DNA SD082.

Clinical background
Lynch syndrome, formerly known as hereditary non-polyposis colorectal cancer (HNPCC), is an adult-onset hereditary cancer susceptibility syndrome predisposing to several cancer types, the most prevalent being colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer and small bowel cancer. It is an autosomal dominantly inherited syndrome that is caused by heterozygous germline mutations in one of the four major DNA mismatch repair genes, i.e. MLH1, MSH2, MSH6 or PMS2. Another cause of Lynch syndrome is deletion of the 3’ part of EPCAM, leading to constitutional epigenetic silencing of the downstream MSH2 gene (Lynch et al. 2015). The estimated contribution of the different genes to Lynch syndrome is 15-40% for MLH1, 20-40% for MSH2, 12-35% for MSH6, 5-25% for PMS2 and <10% for EPCAM . More information about Lynch syndrome is available on http://www.ncbi.nlm.nih.gov/books/NBK1211/.

Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare inherited childhood cancer syndrome characterized by early-onset colorectal cancers, haematological malignancies, and brain tumours. These malignancies are often associated with features of neurofibromatosis type 1 (NF1), such as café-au-lait macules. CMMRD is caused by bi-allelic, i.e. homozygous or compound heterozygous, germline mutations in MLH1, MSH2, MSH6 or PMS2 (Wimmer and Etzler 2008). Mutations in PMS2 are the most common cause of this recessive condition and are responsible for ~50-60% of the CMMRD cases reported thus far (Herkert et al. 2011; Wimmer et al. 2014).

Among the various defects in the PMS2 gene that have been found in Lynch syndrome and CMMRD 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 gene.

Probemix content
The SALSA MLPA Probemix P008-C1 PMS2 contains 47 MLPA probes with amplification products between 128 and 483 nucleotides (nt). This includes 34 probes for the PMS2/PMS2CL genes. In addition, 13 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).

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 and PMS2CL is very consistent among healthy individuals. For this reason, five probes are included that detect sequences present in exons 12-15 of both PMS2 and PMS2CL. As these probes detect both genes, a normal individual has four 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, ten probes are included that detect the copy number of both allelic forms of five SNPs in exons 11-15 of PMS2/PMS2CL. The distribution of these SNP alleles among PMS2 and PMS2CL varies. More information can be found in the product description. 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 gene.

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 (see table below). 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 Reference Selection DNA SD082
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 SD082) is provided with this probemix from MRC Holland. Reference selection DNA SD082 should only be used for initial experiments on DNA samples from healthy individuals with the intention to identify suitable reference samples. SD082 should not be used as a reference sample in subsequent experiments. For further details, consult the Reference Selection DNA SD082 product description, available online: www.mrcholland.com.

Related SALSA MLPA probemixes

Condition		Gene	Probemix
Lynch syndrome (HNPCC)		MLH1	P003, ME011, P248 (confirmation of P003), ME042
		MSH2	P003, ME011, P248 (confirmation of P003)
		MSH6	P072, ME011
		PMS2	P008, ME011
		EPCAM	P003, P072, ME011
Polyposis syndrome	MAP	MUTYH	P378, P043, P072
	AFAP	APC	P043
	FAP	APC	P043

Sample DNA
Sample DNA developed for this product:

• SD082 Reference Selection DNA - included with this probemix.