Intended use: The SALSA MLPA probemix P090 BRCA2 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. This product 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 and not for use with DNA extracted from formalin-fixed paraffin embedded or fresh tumour materials. Deletions or duplications detected with the P090 BRCA2 probemix must be verified by using the SALSA MLPA probemix P077 BRCA2 Confirmation or a different technique. 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.
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: 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 and
BRCA2 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 and
BRCA2 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 and
BRCA2 genes cause around 15% of ovarian cancers overall (Pal et al. 2005).
Deletions or duplications are more frequent for
BRCA1 than for
BRCA2. 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/.
Probemix content: This SALSA MLPA probemix P090-C1 BRCA2 contains 50 MLPA probes with amplification products between 130 and 490 nucleotides (nt). This includes 40 probes for the BRCA2 gene region. In addition, ten 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.
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.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).
| Probemix |
Gene 1 |
Gene 2 |
Coverage |
Use for |
Remarks |
| P045 BRCA2/CHEK2 |
BRCA2 |
CHEK2 |
BRCA2: Each exon.
CHEK2 Exon 1, 9, 1100delC mutation (exon 11) |
Initial testing |
Identical BRCA2 probes as P090 |
| P090 BRCA2 |
BRCA2 |
- |
Each exon |
Initial testing |
Identical BRCA2 probes as P045 |
| P077 BRCA2 Confirmation |
BRCA2 |
- |
Each exon |
Confirmation |
BRCA2 probes target different ligation sites than probes in P090/P045 |
Sample DNA
Sample DNA developed for this product: