The SALSA MLPA Probemix P008 PMS2 is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative assay 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.

For the full intended purpose, see the product description.

Lynch syndrome (LS)

Lynch syndrome (LS), formerly known as hereditary non-polyposis colorectal cancer (HNPCC), is an inherited disorder characterized by an increased predisposition to several cancer types. It is an autosomal dominantly inherited syndrome with gene-dependent, age-related penetrance. Prevalence of LS in the general population has been estimated at 1:279 (Evans et al. 2021, Kunnackal John et al. 2021). LS accounts for 2-3% of all colorectal cancer (CRC) cases, 3% of endometrial cancer (EC) cases and <1% of ovarian cancers. Muir-Torre syndrome (MTS), which is considered a clinical variant of LS, is characterized by synchronous or metachronous occurrence of cutaneous tumours, associated with at least one LS-related internal cancer. LS is caused by heterozygous germline mutations in one of the four major DNA mismatch repair (MMR) genes, i.e. MLH1, MSH2, MSH6 or PMS2. Another cause of LS 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 LS is 15-40% for MLH1, 20-40% for MSH2, 12-35% for MSH6, 5-25% for PMS2, and <10% for EPCAM (GeneReviews). It is estimated that 20-55% of the pathogenic PMS2 mutations identified in LS are attributed to large deletions or duplications encompassing one or more exons. Point mutations and small indels constitute 45-80% of the pathogenic PMS2 mutations (GeneReviews). Tumors exhibit MMR deficiency, which is the consequence of somatic inactivation of the wild-type allele of the affected gene and leads to microsatellite instability (MSI) in the tumor cell DNA, which is the molecular hallmark of the disease.

Constitutional mismatch repair deficiency syndrome

CMMRD is a rare inherited childhood cancer syndrome characterized by early-onset colorectal cancers, hematological malignancies, and brain tumors. These malignancies are often associated with features of neurofibromatosis type 1 (NF1), such as café-au-lait macules (Wimmer and Etzler 2008). CMMRD is a highly penetrant, lethal syndrome with almost 100% mortality by age 35. The syndrome affects 1 in 1,000,000 newborns. 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). Overall, the percentage of CMMRD syndrome cases caused by large deletions in PMS2 is ~12% (Bodo et al. 2015, Herkert et al. 2011). Whereas MMR deficiency is only seen in tumor cells in LS patients, it is seen in all cells of CMMRD patients.

PMS2 and PMS2CL

The PMS2 gene spans 36 kilobases on chromosome 7p22.1 and contains 15 exons. Mutation analysis of the PMS2 gene is complicated by the presence of at least 15 highly homologous non-functional pseudogenes. There are several known pseudogenes present on the long arm of chromosome 7 that have exons 1 to 5 in various organisations, and one highly homologous pseudogene (PMS2CL) which lies in an inverted 100 kb segmental duplication located ~700 kb centromeric of PMS2 (De Vos et al. 2004) [see figure from Lotte]. PMS2CL contains a copy of the 3' end of PMS2, more specifically of exons 9 and 11-15. A region of 2.7 kb encompassing PMS2 exon 10 is absent in PMS2CL (van der Klift et al. 2010). No reliable sequence differences exist between PMS2 exons 12-15 and the associated exons in PMS2CL (van der Klift et al. 2010). As a consequence, many techniques used for copy number determination, including P008 PMS2, cannot discriminate between exons 12-15 of PMS2 and the associated exons in PMS2CL. Only the detection of the combined copy number of the two gene regions is possible, i.e. four copies in healthy individuals. When a copy number change is observed in the PMS2 exons 12-15 region, it can be present in either PMS2 or PMS2CL and follow-up studies are needed to determine which of the two genes is affected. Only copy number changes of PMS2 are linked to LS and CMMRD. No medical consequences stemming from copy number changes of the non-functional PMS2CL pseudogene have been reported to date.

Webinar part 1/3: Why use MLPA for PMS2 CNV detection in Lynch syndrome?

For more information pertaining to the clinical background of LS, CMMRD, and the particularities of the PMS2 gene, please consult the first part of the SALSA MLPA Probemix P008 PMS2 webinar: Part 1: Why use MLPA for PMS2 CNV detection in Lynch syndrome?

Please note: this webinar is the first part in a three-part series.

Due to the presence of the highly similar PMS2CL pseudogene, the distinction between CNVs in PMS2 and PMS2CL is not always clear. However, to obtain as much information about the PMS2 region as possible using the MLPA technique, three different probe types with specific purposes are included in P008 PMS2:

• PMS2-specific probes that target 2 copies in healthy individuals (CN2 probes);
• PMS2/PMS2CL probes that target 4 copies in healthy individuals (CN4 probes);
• PMS2/PMS2CL SNV probe pairs that target both allelic forms of five SNVs present in either exons 11-15 of PMS2 or in the associated exons of PMS2CL.

PMS2-specific (CN2) probes

The PMS2-specific probes target regions throughout exons 1-11 of the PMS2 gene. Although PMS2 exons 9 and 11 share high homology with associated exons in PMS2CL, these probes are specific because they have been specifically designed to detect sequences that are only present in the PMS2 gene. Therefore, the final ratios (FRs) obtained from these probes are directly indicative of the copy number of exons 1-11 of the PMS2 gene.

Combined (CN4) probes for PMS2 and PMS2CL

The combined probes for PMS2 and PMS2CL target regions throughout exons 12-15 of the PMS2 gene and the associated exons in the PMS2CL pseudogene because the very high homology in this region makes design of specific probes impossible. Consequently, the FRs obtained from these probes combine information from the copy number status of both PMS2 and PMS2CL.

PMS2/PMS2CL SNV probe pairs

The PMS2/PMS2CL SNV probe pairs target both allelic forms of five SNVs present in either exons 11-15 of PMS2 or in the associated exons of PMS2CL. The distribution of these SNV alleles among PMS2 and PMS2CL varies. Therefore, the FR obtained from each of these probes correlates to the total number of PMS2 and PMS2CL copies presenting the SNV allele. In individuals without CNVs, the sum of the total number of copies resulting from both allele probes in the probe pair is expected to be 4 (2 alleles in PMS2 and 2 alleles in PMS2CL). This is valid for each probe pair.

Combining information from all probe types

In order to interpret results obtained with P008 PMS2, it may be necessary to combine information from all three probe types described above.

If an aberration is detected using the PMS2-specific (CN2) probes, information from the combined (CN4) probes for PMS2 and PMS2CL and the PMS2/PMS2CL SNV probe pairs can be used to determine the extent of the copy number change. Note that care should be taken if only the exon 11 PMS2-specific (CN2) probe shows an aberration, as gene conversion may occur between PMS2 and PMS2CL in this region.

If an aberration is detected that does not involve the PMS2-specific probes, MLPA alone cannot determine whether the copy number change is present in PMS2 or PMS2CL. Gene-specific long-range PCR and (next generation) sequencing analysis can help determine whether the copy number change is present in the gene or the pseudogene (Li et al. 2015, Vaughn et al. 2011).

Importantly, allocation of the copy number change to either PMS2 or PMS2CL will not be possible when all alleles of PMS2 and PMS2CL share the same variants targeted by the SNV-specific probes. In situations like these, family studies may aid in determining whether the copy number change is present in PMS2 or PMS2CL.

Please note that there are other methods to investigate whether copy number variations are present in PMS2 or PMS2CL, such as capture-NGS. Furthermore, it is important to note that gene conversions are known to occur between PMS2 and PMS2CL, which may involve additional exons and complicate interpretation.

Webinar part 2/3: How to use the different MLPA probes within P008 for PMS2 CNV result interpretation and detection in Lynch syndrome?

For more information pertaining to the use of other techniques to discriminate between PMS2 and PMS2CL, please consult the second part of the SALSA MLPA Probemix P008 PMS2 webinar: Part 2: How to use the different MLPA probes within P008 for PMS2 CNV result interpretation and detection in Lynch syndrome?

Please note: this webinar is the second part in a three-part series.

For data analysis using P008 PMS2, it is critical to select proper reference samples. Suitable reference samples have 2 copies for each allele detected by the ten SNV-specific probes. MRC Holland has selected a cell line with two copies for each of the allelic variants, Reference Selection DNA SD082, which should only be used for the selection of suitable reference samples from your own collection and not as a reference sample. In our experience, approximately one in four samples from healthy individuals tested is suitable as a reference sample. By testing 21 different DNA samples from healthy individuals with P008 PMS2, and including three reactions with Reference Selection DNA SD082, the chance of finding at least three different suitable reference samples is high. Suitable reference samples provide results similar to the Reference Selection DNA SD082 for each probe, including the ten SNV-specific probes.

Webinar part 3/3: Part 3: How to Select Reference Samples by Reference Selection DNA SD082?

For more information about the use of Reference Selection DNA SD082 for suitable reference sample selection, please consult the third part of the SALSA MLPA Probemix P008 PMS2 webinar: Part 3: How to Select Reference Samples by Reference Selection DNA SD082?

Please note: this webinar is the second part in a three-part series.

As a demonstration on how to use Reference Selection DNA SD082, the data made use of throughout part three of the webinar has been made available. Importantly, these instructions must be followed in order to find suitable reference samples for your own experiments. Note that extraction method, tissue type, DNA concentration and treatment must be as similar as possible in all test and reference samples. [JST: this seems out of place.]

[Here will be some links to a page for the demo data or to files or whatever.]

SALSA MLPA P008 PMS2 probemix is CE-marked under the IVDD for in vitro diagnostic (IVD) use in Europe. This assay has also been registered for IVD use in Colombia and Israel.

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

SALSA Reference Selection DNA SD082 can be used to aid in the selection of suitable reference samples for the P008 SALSA MLPA P008 PMS2 probemix probemix. Reference Selection DNA can only be used in initial experiments on DNA samples from healthy individuals from your sample collection with the intention to identify suitable reference samples. SD082 cannot be used as a reference sample in subsequent experiments.

A vial of SALSA Reference Selection DNA SD082 is included with every order of the P008 SALSA MLPA P008 PMS2 probemix 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.

We have no information about specific commercially available positive samples that can be used with this product.

SALSA MLPA probemixes related to Lynch syndrome or polyposis syndrome

Various related products

• ME011 SALSA MLPA ME011 Mismatch Repair genes probemix: Mismatch Repair genes (MMR): methylation profiling of MLH1, MSH2, MSH6 and PMS2.
• ME024 SALSA MLPA ME024 9p21 CDKN2A/2B region probemix: CDKN2A/2B region.
• P003 SALSA MLPA P003 MLH1/MSH2 probemix: HNPCC: primary screening MLH1 and MSH2.
• P043 SALSA MLPA P043 APC probemix: Colon cancer (polyposis), genes included: APC and 3 MUTYH probes.
• P072 SALSA MLPA P072 MSH6-MUTYH probemix: Probes for all MSH6 exons + some EPCAM, MSH2 ex1, MUTYH & MLH1 ex1 probes.
• P248 SALSA MLPA P248 MLH1-MSH2 Confirmation probemix: HNPCC: confirmation of results obtained with P003 (MLH1 and MSH2).
• P378 SALSA MLPA P378 MUTYH probemix: Contains probes for each of the 18 MUTYH exons.

Resources have not been updated in the test environment, but are likely to include the webinar/demo data in some form or another.

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

References have not been updated in the test environment.