Intended purpose
The SALSA MLPA Probemix P225 PTEN is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in PTEN in genomic DNA isolated from human peripheral whole blood specimens. P225 PTEN is intended to confirm a potential cause for and clinical diagnosis of PTEN Hamartoma Tumour Syndrome (PHTS) and for molecular genetic testing of at-risk family members. PHTS includes Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), PTEN-related Proteus syndrome (PS), and Proteus-like syndrome (PLS). This probemix can also be used for the detection of deletions or duplications in the PTEN pseudogene (PTENP1) in a research setting.

Copy number variations (CNVs) detected with P225 PTEN 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 PTEN are point mutations, none of which will be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis.

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, parental evaluation, 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. Only in a research setting can this device be used 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.

¹ 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).
² To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

Clinical background
Phosphatase and tensin homolog (PTEN) is a tumour suppressor gene that is mutated in a large number of cancers at high frequency. Defects in the PTEN gene are the main cause of PTEN Hamartoma Tumour Syndrome (PHTS), which is a dominantly inherited cancer predisposition syndrome, characterized by multiple hamartomas in several areas of the body. PHTS includes Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), PTEN-related Proteus syndrome (PS), and Proteus-like syndrome (PLS) (GeneReviews: https://www.ncbi.nlm.nih.gov/books/NBK1488/).

CS (OMIM #158350) is inherited in an autosomal dominant manner and comprises 85% of PHTS cases. The incidence of CS is estimated to be 1:200,000. Affected individuals usually have macrocephaly, trichilemmomas, and papillomatous papules, and present by their late 20s. The lifetime risk of developing breast cancer is 85%, with an average age of diagnosis between 38 and 46 years. The lifetime risk for thyroid cancer (usually follicular, rarely papillary, but never medullary thyroid cancer) is approximately 35%. The risk for endometrial cancer may approach 28%.

BRRS is inherited in an autosomal dominant manner. It is present at birth and is characterized by macrocephaly, intestinal hamartomatous polyposis, lipomas, pigmented macules of the glans penis, intellectual disability (50% of the cases) and development delay. The risk of developing cancer in BRRS patients with a PTEN pathogenic variant is similar to patients with CS.

PS is a highly variable, severe disorder characterized by progressive segmental or patchy overgrowth of diverse tissues of all germ layers, affecting the skeleton, skin, adipose tissue and central nervous systems. PS is a rare condition with an incidence of less than 1 in 1 million people worldwide and is associated with tumours, pulmonary complications, and deep vein thrombosis.

PLS describes individuals that do not meet the diagnostic criteria of Proteus syndrome, but share many of the characteristic signs and symptoms associated with this condition. Inheritance is autosomal dominant in those with a PTEN mutation.

PTENP1 can regulate cellular levels of PTEN (via binding to mRNAs that target PTEN) and thereby suppresses cell growth. It has been shown that PTENP1 is selectively lost in sporadic colon cancer (Poliseno et al. 2010). A specific PTENP1 deletion (not PTEN) was also demonstrated in human melanoma (Poliseno et al. 2011). Furthermore, the importance of PTENP1 as a tumour suppressor has been recently shown in head and neck squamous cell carcinoma (Liu et al. 2017). PTENP1 copy number detection is of great importance in cancer research, however, the clinical validity of this gene is not yet fully established.

Probemix content
The SALSA MLPA Probemix P225-E1 PTEN contains 49 MLPA probes with amplification products between 130 and 496 nucleotides (nt). This includes 22 probes for the PTEN gene, at least one probe per exon, 10 flanking probes, and two probes for the pseudogene PTENP1. In addition, 15 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.