MRC-Holland MLPA. Description version 23;

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1 SALSA MLPA probemix P087-C1 BRCA1 Lot C & C1-0813: As compared to previous version B1, three reference probes have been replaced and three extra reference probes have been included. The 88 and 96 nt control fragments have been replaced (QDX2). Defects in the BRCA1 gene on human chromosome 17 are an important cause of hereditary breast cancer. Features characteristic of hereditary versus sporadic breast cancer are: younger age at diagnosis, frequent bilateral disease, and more frequent occurrence of disease among men. This probemix contains probes for each exon of the BRCA1 gene, as well as 3 probes for the BRCA2 gene which can help in detection of long deletions/duplications in that gene. For complete analysis of the BRCA2 gene, we recommend the use of the P045 or P090 BRCA2 probemixes. Finally, 10 probes for other human genes located on different chromosomes are included as a reference. In the Netherlands, more than 30% of the BRCA1 related cases of hereditary breast cancer are due to copy number changes of one or more exons of this gene (Petrij-Bosch, A. et al., Nature Genet., 17: , 1997). Estimates in other countries are lower and range from 5 to 15% (Unger, M.A. et al., Am. J. Hum. Genet. 67: , 2000). A wide variety of different exon deletions and duplications have been described. Exon deletions and amplifications will usually not be detected by sequence analysis of the complete BRCA1 gene. Known deletions and amplifications can be easily tested by PCR, but the number of different deletions is becoming prohibitively large. Analysis by MLPA is an easy to perform alternative that is also capable of detecting new deletions and amplifications. SALSA probemix P087 BRCA1 has been developed in order to allow rapid confirmation of results obtained with the P002 probemix. We recommend using the P002 probemix for the primary screening. Similar to the P002 probemix, P087 BRCA1 contains one probe for each BRCA1 exon. Distance between the ligation sites of the P087 probes and the corresponding P002 probes is at least 20 nucleotides, with the exception of the exon 24 probe. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the BRCA1 gene in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA test. SALSA probemixes and reagents are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA test probemixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA probemix and reagents requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in Nucleic Acid Research 30, e57 (2002). Related SALSA probemixes P002 BRCA1: Hereditary breast cancer; primary screening BRCA1. P239 BRCA1 region: BRCA1 deletions/duplications in the exon 1 / BRCA1P1/NBR1 region and the BRCA1 upstream region. Also contains some PALB2, BRIP1, ATM and CHEK2 probes. P045 BRCA2/ CHEK2: Hereditary breast cancer, BRCA2 and CHEK2. P090 BRCA2: Identical to P045 BRCA2/CHEK2, but does not contain probes for CHEK2. P077 BRCA2: Results obtained with P045 or P090 can be confirmed with this P077 probemix. P190 CHEK2: Breast cancer susceptibility, genes included: CHEK2, ATM, BRCA1, PTEN, TP53 P057 FANCD2/PALB2: Mutations in PALB2 have been linked to a higher risk on breast cancer. P240 BRIP1/CHEK1: Mutations in BRIP1 have been linked to a higher risk on breast cancer. P041/P042 ATM: Mutations in ATM have been linked to a higher risk on breast cancer. SALSA probemix P087 BRCA1 Page 1 of 5

2 More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands References of SALSA probemix BRCA1 P002/P087 Please note that a PubMed search on BRCA1 and MLPA will show more than 40 articles. Only a selection of the articles published until 2007 is shown. Lim Y. et al. (2007). Identification of novel BRCA large genomic rearrangements in Singapore Asian breast and ovarian patients with cancer. Clin Genet Apr;71(4): Gutierrez-Enriquez S. et al. (2006). Screening for large rearrangements of the BRCA2 gene in Spanish families with breast/ovarian cancer. Breast Cancer Res Treat Oct 25. Preisler S. et al. (2006). Gross rearrangements in BRCA1 but not BRCA2 play a notable role in predisposition to breast and ovarian cancer in high-risk families of German origin. Cancer Genet Cytogenet Jul 1;168(1):44-9. Thomassen M. et al. (2006). Low frequency of large genomic rearrangements of BRCA1 and BRCA2 in western Denmark. Cancer Genet Cytogenet Jul 15;168(2): Indraccolo S. et al. (2006). Establishment and characterization of xenografts and cancer cell cultures derived from BRCA1 -/- epithelial ovarian cancers. Eur J Cancer Jul;42(10): De la Hoya M. Et al. (2006). Genomic Rearrangements at the BRCA1 Locus in Spanish Families with Breast/Ovarian Cancer. Clin Chem Jun 22. Woodward AM, et al. (2005). Large genomic rearrangements of both BRCA2 and BRCA1 are a feature of the inherited breast/ovarian cancer phenotype in selected families. J Med Genet. 42(5):e31. Hogervorst et al (2003). Large genomic deletions and duplications in the BRCA1 gene identified by a novel quantitative method. Cancer Research 63, Montagna et al (2003). Genomic rearrangements account for more than one-third of the BRCA1 mutations in northern Italian breast/ovarian cancer families. Human Molec Genet. 12, Hartmann C et al. (2004). Large BRCA1 gene deletions are found in 3% of German high-risk breast cancer families. Hum Mutat; 24(6):534. Belogianni I et al. Characterization of a novel large deletion and single point mutations in the BRCA1 gene in a Greek cohort of families with suspected hereditary breast cancer. BMC Cancer. 7;4(1):61. Bunyan DJ et al. (2004). Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification. Br J Cancer. 13;91(6): Data analysis The P087-C1 BRCA1 probemix contains 39 MLPA probes with amplification products between 130 and 454 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA denaturation control fragments (Dfragments) at nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix can first be normalised intra-sample by dividing the peak height of each probe s amplification product by the total peak height of only the reference probes in this probemix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised probe ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference probes. Data normalisation should be performed within one experiment. Only samples purified by the same method should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern blotting, long range PCR, qpcr, FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P087 BRCA1 Page 2 of 5

3 Table 1. SALSA MLPA P087-C1 BRCA1 probemix Chromosomal position SALSA MLPA probe reference BRCA1 BRCA Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 * Reference probe L q BRCA2 probe L01774 Exon BRCA1 probe L01268 Exon 1a 154 * Reference probe L p BRCA1 probe L25904 Upstream 167 BRCA1 probe L02239 Exon BRCA1 probe L02240 Exon BRCA1 probe L02254 Exon Reference probe L q BRCA1 probe L12189 Exon BRCA1 probe L02242 Exon BRCA1 probe L02243 Exon BRCA1 probe L02244 Exon BRCA1 probe L02245 Exon * Reference probe L q BRCA1 probe L25905 Exon BRCA2 probe L01770 Exon BRCA1 probe L12190 Exon * Reference probe L q BRCA1 probe L02247 Exon BRCA1 probe L02248 Exon BRCA1 probe L03337 Exon BRCA2 probe L10644 Exon Reference probe L p BRCA1 probe L02250 Exon BRCA1 probe L02251 Exon BRCA1 probe L12877 Exon BRCA1 probe L03285 Exon BRCA1 probe L02255 Exon * Reference probe L q Reference probe L q BRCA1 probe L02256 Exon BRCA1 probe L02257 Exon BRCA1 probe L13116 Exon BRCA1 probe L02259 Exon BRCA1 probe L04795 Exon BRCA1 probe L02537 Exon 1a 445 * Reference probe L p Reference probe L q34 * New in version C1 (from lot C onwards) Changed in version C1 (from lot C onwards). Small change in length or peak height. No change in sequence detected. Note: The BRCA1 exon numbering used is different as compared to the exon numbering used by the NCBI in the NM_ reference sequence! We used the same exon numbering as in all previous versions of this product description. Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. Please note that two probes for exon 13 are present. The 250 nt probe (03411-L25905) detects the same sequence as the exon 13 probe at 295 nt in the SALSA MLPA P002-C2 probemix. SALSA probemix P087 BRCA1 Page 3 of 5

4 Table 2. BRCA1 probes arranged according to chromosomal location SALSA MLPA probe BRCA1 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 2) L25904 Upstream 703 nt before ex 1a, rev TCTGCGCACTCG-TAGTTCCACCCC 0.7 kb L01268 Exon 1a 67 nt before ex 1a TCATCCGGGGGC-AGACTGGGTGGC 0.1 kb L02537 Exon 1a CGTGGCAACGGA-AAAGCGCGGGAA 1.4 kb L02239 Exon , rev GATGGGACACTC-TAAGATTTTCTG 8.3 kb L02240 Exon , rev ACTTACTTGCAA-AATATGTGGTCA 9.3 kb L12877 Exon 5 3 nt after ex 5, rev ACCAAATTATAT-ACCTTTTGGTTA 1.6 kb L02242 Exon TGCTTTTCAGCT-TGACACAGGTTT 0.7 kb L02243 Exon , rev GTAGCCCATACT-TTGGATGATAGA 4.3 kb L02244 Exon 8 9 nt before ex 8 GTTCTTTACCAT-ACTGTTTAGCAG 2.6 kb L02245 Exon 9 10 nt after ex 9, rev CAAAGGTTCTCT-TTGACTCACCTG 1.4 kb L03285 Exon , rev TGCAGAATCCAA-ACTGATTTCATC 2.4 kb L02247 Exon CCTACAACTCAT-GGAAGGTAAAGA 2.5 kb L02248 Exon , rev GGTTAAAATGTC-ACTCTGAGAGGA 8.4 kb L25905 Exon AATGGCTGAACT-AGAAGCTGTGTT 0.1 kb L03337 Exon AAGTGACTCTTC-TGCCCTTGAGGA 5.9 kb L12190 Exon GGCCTTTCTGCT-GACAAGTTTGAG 2.2 kb L02250 Exon CAACAGCTGGAA-GAGTCTGGGCCA 3.2 kb L02251 Exon CCAGAGTCAGCT-CGTGTTGGCAAC 3.5 kb L13116 Exon 17 9 nt after ex 17, rev TGTAAAGGTTCT-TGGTATACCTGT 3.7 kb L02254 Exon GGAAAATGGGTA-GTTAGCTATTTC 0.5 kb L02255 Exon nt before ex 19 TGAAAAGAGCAC-GTTCTTCTGCTG 6.3 kb L02256 Exon nt before ex 20 TTTCTCTTATCC-TGATGGGTTGTG 6.1 kb L02257 Exon nt after ex 21 CCTTTGTCTTAC-ATAGTGGAGTAT 1.8 kb L12189 Exon , rev TGTACCATCCAT-TCCAGTTGATCT 1.6 kb L02259 Exon nt after ex 23 CTGCATGTACCT-GTGCTATATGGG 1.8 kb L04795 Exon ATGTGTGAGGCA-CCTGTGGTGACC stop codon (ex 24) Small change in length or peak height as compared to previous versions. No change in sequence detected. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Note: The exon numbering used is different as compared to the exon numbering used by the NCBI in the NM_ reference sequence! We used the same exon numbering as in all previous versions of this product description. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. BRCA1 exon 4 does not exist in the exon numbering that we used. Exon 11 is longer than all the other exons combined. A pseudogene containing a very similar copy of exons 1a and 2 is present on a short distance from the BRCA1 gene. The two MLPA probes for these exons should not generate a signal on this pseudogene as these probes have a mismatch at the probe ligation site when annealed to these pseudogene sequences. Relative probe signals for these two exons are higher than for most other probes. The probe signals of these two probes tend to be reduced when large amounts of DNA is used as compared to reference reactions, and when PCR inhibitors are present in the DNA samples. If samples appear to have a deletion of these two exons, we recommend repeating the test with lower amounts of DNA, e.g. 40 ng. SALSA probemix P087 BRCA1 Page 4 of 5

5 Table 3. BRCA2 probes arranged according to chromosomal location SALSA MLPA probe BRCA2 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 2) L01774 Exon 1 At mrna start CAGCGCGGGCTT- GTGGCGCGAGCT 10.6 kb L10644 Exon TGTAACACCACA- AAGAGATAAGTC 10.2 kb L01770 Exon TCTGAAGAACCA- ACTTTGTCCTTA stop codon (ex 27) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. SALSA MLPA probemix P087-C1 BRCA1 sample picture D ye S ignal Size Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P087-C1 BRCA1 (lot C1-0114). Implemented Changes compared to the previous product description version(s). Version 23 (53) - Product description adapted to a new lot (lot number added, new picture included). Version 22 (51) - Product description adapted to a new version (lot number added, new picture included). Version 21 (48) - Warning added in Table 1, 130 nt probe L Version 20 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 19 (48) - Various minor textual changes on page 1. - Ligation sites of the probes targeting the BRCA1 gene updated according to new version of the NM_reference sequence. - Remark on RefSeqGene standard and transcript variant added below Table 2. Version 18 (46) - Mistake in Table 2 corrected (partial sequences of 157 nt and 148 nt probes were mixed up). SALSA probemix P087 BRCA1 Page 5 of 5