Intended use: The SALSA MLPA probemix P056 TP53 is an in vitro diagnostic (IVD)1 or a research use only (RUO) assay for the detection of deletions or duplications in the human TP53 gene in order to confirm a potential cause and clinical diagnosis for Li-Fraumeni syndrome (LFS1) or Li-Fraumeni-like syndrome (LFL). In addition, this assay can be used to detect deletions or duplications in the human CHEK2 gene exons 8, 10 and 13 and the CHEK2 1100delC mutation, to determine a suggested cause for Li-Fraumeni syndrome (LFS2) in a research setting. This assay 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. In a research setting this assay can be used on DNA derived from fresh or formalin-fixed paraffin-embedded tumour tissue. Most defects in the TP53 gene are point mutations, the majority of which are not detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of this gene. Deletions or duplications detected with the P056 TP53 probemix should be verified by another technique. In particular, deletions or duplications detected by only a single probe always require validation by another method. This probemix is not intended to be used as a standalone assay for clinical decisions. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.
1Please 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).
Clinical background: Li-Fraumeni syndrome (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome. LFS1 is characterised by autosomal dominant inheritance and early onset of tumours, multiple tumours within an individual, and multiple affected family members. The most common types of tumours are soft tissue sarcomas and osteosarcomas, pre-menopausal breast cancer, brain tumours, leukaemia, and adrenocortical carcinoma. Approximately 70% of LFS1 cases contain germline mutations in the tumour suppressor gene TP53. Families presenting incomplete features of LFS are referred as having Li-Fraumeni-like syndrome (LFL) and around 20-40% of these patients have a germline mutation in TP53 (Ruijs et al. 2010). Somatic pathogenic variants in TP53 are found in about 50% of all tumours, making it one of the most frequently altered genes in human cancers. Around 50% of the individuals carrying germline mutations in TP53 will develop cancer by the age of 30 years with a lifetime risk of up to 70% in men and almost 100% in women. There is no definitive information on the optimal methods and efficacy of tumour surveillance for children or adults with a germline TP53 pathogenic variant. For more information: https://www.ncbi.nlm.nih.gov/books/NBK1311/.
A second form of Li-Fraumeni syndrome (LFS2) is caused by mutations in the CHEK2 gene. In particular, the CHEK2 1100delC mutation has been suggested as an underlying cause, though also a deletion in CHEK2 was found in a patient fulfilling LFL criteria (Ruijs et al. 2009). Researchers are uncertain whether CHEK2 gene mutations actually cause LFS or are merely associated with an increased risk of several types of cancer, including those cancers often seen in LFS. For more information: https://omim.org/entry/609265.
Probemix content: This SALSA MLPA probemix P056 TP53 contains 40 probes with amplification products between 129 and 490 nt (Table 1), including 14 reference probes. At least one probe is present for each exon of transcript variant 1 of TP53; two probes are present for exon 1 and exon 4b. One probe is included for exon 9a, which is present in transcript variant 3. Several flanking probes for TP53 are present to determine the extent of the deletion/duplication. In addition, there are four probes included for CHEK2: a probe for exon 8, 10 and 13 and a mutation specific probe for the CHEK2 1100delC mutation, which will only generate a signal when the mutation is present. The identity of the genes detected by the reference probes is available online (www.mlpa.com) and in Table 2c.
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 in product description). 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 Binning DNA SD067: The SD067 Binning DNA provided with this probemix can be used as Binning DNA sample for binning of the CHEK2 1100delC mutation-specific probe (208 nt probe, 18318-L26751). SD067 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD067 Binning DNA 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), as for this purpose true mutation positive patient samples or cell lines should be used. It is strongly advised to use DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For further details, please consult the SD067 Binning DNA product description provided online at www.mlpa.com.
Sample DNA developed for this product: