MRC-Holland MLPA. Description version 31;

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1 SALSA MLPA probemix P002-C2 BRCA1 Lot C2-0313, C2-0113, C2-0811, C As compared to the previous C1 version (lot 1209 & 0409), the 88 and 96 nt DNA Denaturation 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 P002-C2 BRCA1 probemix contains probes for each exon of the BRCA1 gene. In addition, it contains 9 reference probes for other human genes located on different chromosomes. 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., 1997, Nat Genet.). The majority of these are due to two frequently occurring founder mutations: deletion of exon 13 or exon 22. Estimates in other countries are lower and range from 5 to 15% (Unger, M. A. et al., 2000, Am J Hum Genet.). 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. 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 area 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 area, 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. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. 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 this 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 P087 BRCA1: Results obtained with P002 can be confirmed with this probemix. P239 BRCA1 region: Characterization of BRCA1 deletions/duplications. 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/P090 can be confirmed with this 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. More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA probemix P002 BRCA1 Page 1 of 6

2 References for SALSA probemix P002 BRCA1 A PubMed search on BRCA1 AND (MLPA or ligation dependent probe) will generate more than 75 hits, most of which deal with the use of this P002 product for the detection of large rearrangements in BRCA1. Data analysis The P002-C2 probemix contains 35 MLPA probes with amplification products between 127 and 463 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 area of each probe s amplification product by the total area 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 by. Info/remarks/suggestions for improvement: info@mlpa.com. Please note BRCA1 exon 4 does not exist. Two alternative exons 1 exist: 1a and 1b. Probes for both exons 1a as well as 1b are included. Exon 1b is not present in Genbank mrna sequence U14680 and NM_ Apparent deletions of exons 1a + 1b + 2 should be treated with caution, as contamination in the sample may prevent complete denaturation of the CpG islands near exons 1a, 1b and 2, resulting in lower signals for these probes. Exon 11 is longer than all the other exons combined. We therefore made two probes of exon 11, at a distance of 2800 nt from each other. A pseudogene (BRCA1P1) containing a very similar copy of exons 1a, 1b and 2 is present on a short distance from the BRCA1 gene. The three 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. The probe signals of these three probes tend to be reduced when large amounts of DNA is used as compared to reference samples, or when PCR inhibitors are present in the DNA samples. If samples appear to have a deletion of these three exons, we recommend repeating the test with lower amounts of DNA, e.g. 40 ng. SALSA probemix P002 BRCA1 Page 2 of 6

3 Table 1. SALSA MLPA P002-C2 BRCA1 probemix Length (nt) SALSA MLPA probe Chromosomal position reference 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 127 Reference probe L q Reference probe L p BRCA1 probe L00268 exon 1a 157 BRCA1 probe L00269 exon 1b 166 BRCA1 probe L00270 exon BRCA1 probe L00341 exon BRCA1 probe L00272 exon Reference probe L q BRCA1 probe L00342 exon BRCA1 probe L00274 exon BRCA1 probe L00569 exon BRCA1 probe L00581 exon BRCA1 probe L00277 exon Reference probe L q BRCA1 probe L00345 exon BRCA1 probe L00279 exon BRCA1 probe L00280 exon ± BRCA1 probe L02074 exon BRCA1 probe L00349 exon Reference probe L p BRCA1 probe L00347 exon BRCA1 probe L00003 exon BRCA1 probe L00283 exon ± BRCA1 probe L00284 exon BRCA1 probe L00285 exon Reference probe L q BRCA1 probe L00356 exon BRCA1 probe L12004 exon BRCA1 probe L00288 exon BRCA1 probe L00289 exon ± BRCA1 probe L13862 exon Reference probe L p Reference probe L q Reference probe L p ± BRCA1 probe L12001 exon 13 ± Probe has a higher standard deviation. Note: The exon numbering in Table 1 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. SALSA probemix P002 BRCA1 Page 3 of 6

4 Table 2. BRCA1 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe BRCA1 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 2) L00268 exon 1a reverse AGCAGAGGGTGA-AGGCCTCCTGAG 0.2 kb L00269 exon 1b 208 nt after exon 1a AGGGGGCACTGA-GTGTCCGTGGGG 1.0 kb L00270 exon ATTTATCTGCTC-TTCGCGTTGAAG 8.3 kb L00341 exon TCAAGGAACCTG-TCTCCACAAAGT 9.3 kb L00272 exon ACTTCTCAACCA-GAAGAAAGGGCC 1.6 kb L00342 exon CGAGATTTAGTC-AACTTGTTGAAG 0.8 kb L00274 exon AACCGTGCCAAA-AGACTTCTACAG 4.3 kb L00569 exon CTTGGAACTGTG-AGAACTCTGAGG 2.6 kb L00581 exon CGTTAATAAGGC-AACTTATTGCAG 1.3 kb L00277 exon TTGTTACAAATC-ACCCCTCAAGGA 1.1 kb L00345 exon AAGCGTGCAGCT-GAGAGGCATCCA 2.8 kb L00279 exon TCCTAGCCCTTT-CACCCATACACA 1.0 kb L00280 exon CTCTGAAGACTG-CTCAGGGCTATC 8.5 kb 295 ± L02074 exon AATGGCTGAACT-AGAAGCTGTGTT 0.3 kb 463 ± L12001 exon nt after exon 13 CTCACAACTAAT-ATACCAGTCAGA 5.7 kb L00349 exon CCAGAAGGCCTT-TCTGCTGACAAG 2.1 kb L00347 exon CTCTGGGAGTCT-TCAGAATAGAAA 3.2 kb L00003 exon ATCTGGAATCAG-CCTCTTCTCTGA 3.5 kb L00283 exon TTGCCAGAAAAC-ACCACATCACTT 3.7 kb 355 ± L00284 exon TTTGTGTGTGAA-CGGACACTGAAA 0.6 kb L00285 exon ACCCAGTCTATT-AAAGAAAGAAAA 6.3 kb L00356 exon TGGTCAATGGAA-GAAACCACCAAG 6.0 kb L12004 exon GAAATCTGTTGC-TATGGGCCCTTC 1.9 kb L00288 exon TTCTGTGGTGAA-GGAGCTTTCATC 1.5 kb L00289 exon TCCACCCAATTG-TGGTTGTGCAGC 2.5 kb 427 ± L13862 exon TCAATGGAAGGA-GAGTGCTTGGGA stop codon (ex 24) The NM_ sequence represents transcript variant 1 and is a reference sequence in the RefSeqGene project. ± Probe has a higher standard deviation. Different Genbank #: L Either exon 1a or 1b is used in transcripts. Please note that the ligation sites mentioned are the locations in the Genbank mrna reference sequence NM_ , not the location from the start codon. We have been informed by Claire Morgan (Birmingham) that an apparent deletion of exon 19 (364 nt) proved to be due to the presence of the c.5276g>t (pval1719val, rs ) polymorphism. The 295 nt exon 13 probe detects the same sequence as the 244 nt probe in SALSA MLPA P087 probemix. Note: The exon numbering in Table 2 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. SALSA probemix P002 BRCA1 Page 4 of 6

5 SALSA MLPA probemix P002-C2 BRCA1 sample picture D ye S ign al Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P002-C2 BRCA1 (lot C2-0313). Implemented Changes the following has been altered compared to the previous product description version(s). Version 31 (50) - Textual changes below Table 1 and 2. Version 30 (50) - Product description adapted to a new product lot (lot number added, new picture included). Version 29 (49) - Product description adapted to a new product lot (lot number added, new pictures included). Version 28 (47) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 27 (47) - Product description adapted to a new product lot (lot number added, new picture included). - Warning on primer dimer formation when using the old PCR protocol removed from page 2. Version 26 (46) - Warning changed on page 2. Use new PCR primer mix to avoid primer-dimer formation. Version 25 (46) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). Version 24 (45) - Unclear remark probe 136 nt removed. Version 23 (45) - Warning added in Table 1, 136 nt probe L05978, 295 nt probe L02074, 355 nt probe L00284, and 463 nt probe L New reference added on page 2. SALSA probemix P002 BRCA1 Page 5 of 6

6 Version 22 (45) - Product description adapted to a new lot (lot number added, new picture included). - Ligation sites updated according to new version of the NM_reference sequence. - Warning added below Tables 1 and 2 that the exon numbering used is different from the NCBI exon numbering in the NM_ reference sequences. Version 21 (45) - Information about the old version of P002-B1 has been removed from page 1. - Various minor textual changes on page 1; data analysis has been modified; tables have been numbered. SALSA probemix P002 BRCA1 Page 6 of 6