The SALSA MLPA probemix P045 BRCA2/CHEK2 is an in vitro diagnostic (IVD)1
or a research use only (RUO) assay for the detection of deletions or duplications in the human BRCA2
gene, and the presence of the c.156_157insAlu mutation, in order to confirm a potential cause and clinical diagnosis for hereditary breast and ovarian cancer (HBOC) or in rare cases Fanconi Anemia type D1. In addition, the 1100delC mutation and deletions or duplications of exon 1 and 9 in CHEK2
can be detected, in order to determine predisposition to breast cancer, but also other cancer types, included but not limited to colon and prostate cancer. This product can also be used for molecular genetic testing of family members who are at risk for the mentioned aberrations.
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 P045 BRCA2/CHEK2 probemix must be verified by using the SALSA MLPA probemix P077 BRCA2 Confirmation or a different technique. P077 BRCA2 Confirmation cannot be used to verify CHEK2
mutations. However, the P190 CHEK2 probemix is available for deletion or duplication analysis of other CHEK2
exons. Most defects in the BRCA2
gene are point mutations, the majority of which will not be detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of the BRCA2
gene. This assay 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.
Please 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).
Breast and ovarian carcinomas are among the most common malignancies in developed countries. The majority of cases are considered sporadic, but in a substantial portion, a clear history of cases within a family is present. The BRCA1 and BRCA2 proteins are associated with the activation of double-strand break repair and homologous recombination and are important in maintaining genomic stability. Germline mutations in the BRCA1
genes are linked to a high risk of young-onset hereditary breast and ovarian cancer. Features characteristic of hereditary, versus sporadic, breast cancer are: younger age at diagnosis, frequent bilateral disease, and more frequent occurrence of disease among male relatives. Mutations in the BRCA1
genes account for about 20 to 25% of hereditary breast cancers (Easton 1999) and about 5 to 10% of all breast cancers (Campeau et al. 2008). In addition, mutations in the BRCA1
genes cause around 15% of ovarian cancers overall (Pal et al. 2005).
Deletions or duplications are more frequent for BRCA1
than for BRCA2
in most populations.
The prevalence of deletions or duplications is dependent on the studied population and ranges from 0% to 11% of all BRCA2
mutations (Agata et al. 2005, Woodward et al. 2005, Casilli et al. 2006, Stadler et al. 2010).
More information is available at http://www.ncbi.nlm.nih.gov/books/NBK1247/
Biallelic pathogenic variants of BRCA2
can result in Fanconi Anemia (FA) type D1. FA is characterized by physical abnormalities (such as short stature or abnormal skin pigmentation), bone marrow failure and increased risk for malignancies. The incidence of FA in general is 1:160,000, of which type D1 comprises around 3% of the cases. FA type D1 is associated with early-onset acute leukaemia and solid tumours. More information on FA is available at https://www.ncbi.nlm.nih.gov/books/NBK1401/
mutations are most frequently found, but other genes are also associated with an increased risk for developing breast and ovarian cancer, including CHEK2
. The protein CHK2 is a cell cycle checkpoint regulator and a putative tumour suppressor. In non-BRCA1/2 breast cancer families, patients heterozygous for the 1100delC mutation have a two times increased risk of developing breast cancer and have a higher contralateral breast cancer rate (Huijts et al. 2014, Kriege et al. 2014). A deletion of exon 9 and 10 in CHEK2
has been found mainly in Slavic populations and is associated with a two times higher risk for breast cancer (Walsh et al. 2006).
The SALSA MLPA Probemix P045-D1 BRCA2/CHEK2 contains 51 MLPA probes with amplification products between 130 and 500 nucleotides (nt). This includes 40 probes for the BRCA2
region and three probes for the CHEK2
region. In addition, eight 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.mlpa.com
At least one MLPA probe is present for each exon in the BRCA2
transcript; two probes are present for exons 1 and 3, three probes are present for exons 10 and 27, and six probes are present for exon 11. One of the probes for exon 3 detects the wild type sequence of the c.156_157insAlu mutation and a reduced signal can point towards the presence of this mutation or
a deletion of exon 3. In addition, there is a probe for a sequence upstream and a probe for a sequence downstream of BRCA2
For the CHEK2
gene, one probe is present for exons 1 and 9. Moreover, one probe specific for the CHEK2
1100delC mutation is included, which will only generate a signal when the mutation is present.
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 Binning DNA SD067:
The SD067 Binning DNA provided with this probemix can be used for binning of the CHEK2
1100delC mutation-specific probe (490 nt probe 01772-L01336). 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/SNP positive patient samples or cell lines should be used. It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD067 Binning DNA product description, available online: www.mlpa.com
SALSA Artificial Duplication DNA SD024:
In case no positive DNA sample is available in your laboratory, an artificial duplication DNA sample for this probemix (catalogue number SD024) can be ordered from MRC-Holland. This SD024 Artificial Duplication DNA will show a duplication of two or more probes when using the following probemixes: P045, P090 and P077 for BRCA2
and P002 and P087 for BRCA1.
The SD024 Artificial Duplication DNA is a mixture of human female genomic DNA and a titrated amount of plasmid containing selected probe target sequences. For further details, please consult the SD024 Artificial Duplication DNA product description, available online: www.mlpa.com
. This product is for research use only (RUO).
||BRCA2: Each exon.
CHEK2 Exon 1, 9, 1100delC mutation (exon 11)
||Identical BRCA2 probes as P090
||Identical BRCA2 probes as P045
|P077 BRCA2 Confirmation
||BRCA2 probes target different ligation sites than probes in P090/P045
Sample DNA developed for this product: