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SALSA MLPA Probemix P045 BRCA2/CHEK2

Breast and ovarian cancer syndrome, hereditary (HBOC); Fanconi anemia type D1; Susceptibility to breast cancer; Susceptibility to other cancer types

Region: BRCA2 13q13.1; CHEK2 22q12.1

MLPA | CE CO CR IL
Intended purpose
The SALSA MLPA Probemix P045 BRCA2/CHEK2 is an in vitro diagnostic (IVD)1 or a research use only (RUO) semi-quantitative assay2 for the detection of deletions or duplications in the BRCA2 gene and the presence of the wildtype sequence of the BRCA2 c.156_157insAlu mutation in genomic DNA isolated from human peripheral whole blood specimens. P045 BRCA2/CHEK2 is intended to confirm a potential cause for and clinical diagnosis of hereditary breast and ovarian cancer (HBOC) syndrome, and, in rare cases, Fanconi Anemia type D1. In addition, deletions and duplications of CHEK2 exon 1 and exon 9 as well as the presence of the CHEK2 c.1100delC mutation can be detected with this probemix in order to confirm a potential cause for breast cancer and other CHEK2-related cancer types. This product can also be used for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P045 BRCA2/CHEK2 should be confirmed with 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 all CHEK2 exons. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in the BRCA2 and CHEK2 genes are point mutations, the majority of which will not 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, 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, e.g from DNA extracted from formalin-fixed paraffin embedded (FFPE) or fresh tumour materials.

There are three probemixes available for BRCA2 testing at MRC Holland. Content and use is described below:

SALSA MLPA Probemix Target gene(s) Coverage Use for Remarks
P045 BRCA2/CHEK2 BRCA2, CHECK BRCA2: Each exon
CHEK2: Exon 1, 9, 1100delC mutation
(exon 11)
Initial testing by MLPA All BRCA2 probes are identical to those present in P090 BRCA2
P090 BRCA2 BRCA2 BRCA2: Each exon Initial testing by MLPA All BRCA2 probes are identical to those present in P045 BRCA2/CHEK2
P077 BRCA2 Confirmation BRCA2 BRCA2: Each exon Confirmation of MLPA results The ligation sites of all P077 BRCA2 probes differ from those targeted by P045/P090 BRCA2

1 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).
2 To be used in combination with a SALSA MLPA Reagent Kit, SALSA Binning DNA SD067 and Coffalyser.Net analysis software.

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 for hereditary, versus sporadic, breast cancer are: younger age at diagnosis, frequent bilateral disease, and more frequent occurrence of diseases such as prostate and breast cancer among male relatives. Mutations in the BRCA1 and BRCA2 genes account for about 20-25% of hereditary breast cancers (Easton 1999) and about 5-0% of all breast cancers (Campeau et al. 2008). In addition, mutations in the BRCA1 and BRCA2 genes cause around 15% of ovarian cancers (Pal et al. 2005). Women with a germline BRCA2 mutation have a 45-62% lifetime risk of developing breast cancer, while the risk of women in the general population is 12%. The lifetime risk of developing ovarian cancer in women with a germline BRCA2 mutation is 11-17%, compared to 1-2% in the general population. Deletions and duplications are more frequent for BRCA1 than for BRCA2 in most populations. CNVs in BRCA2 account for 2-3% of all pathogenic BRCA2 mutations, dependent on the population. 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/.

BRCA1 and BRCA2 mutations are the most frequent aberrations 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 CHEK2 c.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).

Probemix content
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. 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 wildtype sequence of the c.156_157insAlu mutation and a reduced signal can point towards the presence of this mutation or a (partial) deletion of exon 3. In addition, a probe detecting a sequence upstream and a probe detecting a sequence downstream of the BRCA2 gene are present to determine the extent of a deletion or duplication.

For the CHEK2 gene, one probe is present for exons 1 and 9. Moreover, one probe specific for the CHEK2 c.1100delC mutation is included, which will only generate a signal when the mutation is present.

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.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.

SALSA Binning DNA SD067
The SD067 Binning DNA provided with this probemix can be used for binning of all probes including the CHEK2 c.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). 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.mrcholland.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 BRCA2; P002 and P087 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.mrcholland.com. This product is for research use only (RUO).

Sample DNA
Sample DNA developed for this product:

Order Items

Probemix

Item no.
Description
Technology
Price
P045-025R
SALSA MLPA Probemix P045 BRCA2/CHEK2 – 25 rxn
€ 260.00
P045-050R
SALSA MLPA Probemix P045 BRCA2/CHEK2 – 50 rxn
€ 510.00
P045-100R
SALSA MLPA Probemix P045 BRCA2/CHEK2 – 100 rxn
€ 998.00

Required Reagents (Sold Separately)

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA Reagent Kit – 100 rxn – FAM
€ 316.00
EK1-Cy5
SALSA MLPA Reagent Kit – 100 rxn – Cy5
€ 316.00
EK5-FAM
SALSA MLPA Reagent Kit – 500 rxn – FAM
€ 1455.00
EK5-Cy5
SALSA MLPA Reagent Kit – 500 rxn – Cy5
€ 1455.00
EK20-FAM
SALSA MLPA Reagent Kit – 2000 rxn – FAM
€ 5590.00

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CE-marked products are for In Vitro Diagnostic (IVD) use only in EU (candidate) member states and members of the European Free Trade Association (EFTA), and the UK.

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