SALSA MLPA probemix P045-B3 BRCA2/CHEK2 Lot B3-0715, B3-0714, B3-1113, B3-0413, lot B3-0711. As compared to version B2 (lot B2-0410), the 88 and 96 nt control fragments have been replaced (QDX2). Breast carcinoma is the most common malignancy among women in developed countries and family history remains the strongest single predictor of breast cancer risk. The BRCA1 and BRCA2 proteins are associated with the activation of double-strand break repair and/or homologous recombination and are important in maintaining genomic stability. Mutations in BRCA1 and BRCA2 genes account for about 16% of hereditary breast cancers and about 2 to 4% of all breast cancers. In addition, mutations in BRCA1 and BRCA2 genes account for around 15% of ovarian cancers overall. BRCA2 mutations are less frequent than BRCA1 mutations but in families with male breast cancer cases, BRCA2 mutations may be more frequent. The P045-B3 probemix contains probes for all exons of the BRCA2 gene (13q12.3). Two probes are present for exons 1, 3 and 27, and for the large exon 11. As reference, eight probes for other human genes located on different chromosomes are included. In addition, two probes are present for sequences just before and after the BRCA2 gene. Furthermore, three probes for the CHEK2 gene on 22q12.1 are included. One of these probes will only result in an amplification product in case the DNA sample contains the CHEK2 1100delC mutation. The 1100delC allele appears to result in an increased risk for breast cancer and prostate cancer. The 1100delC allele has been found in the Netherlands in 1.1% of healthy individuals and in 5.1% of individuals with breast cancer, including 13.5% of individuals from families with male breast cancer. Next to this P045 BRCA2 probemix, two other BRCA2 specific MLPA probemixes are available. The P090 probemix is almost identical to P045 but does not contain any CHEK2 probes. The P077 BRCA2 probemix contains different probes for this gene and can be used for a first rapid confirmation of results obtained with P045 or P090. SD029 Sample DNA Please note that the SNP-specific probe has only been tested on control plasmids and not on positive human DNA samples with the CHEK2 1100delC mutation! This SD029 sample DNA is provided with each probemix vial and can be used in data binning in the fragment analysis and as a positive control for the mutationspecific probe(s) (see next page). This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the BRCA2 and CHEK2 genes 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 MLPA 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). More information Website : www.mlpa.com E-mail : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA P045 BRCA2/CHEK2 probemix Page 1 of 8
Related SALSA probemixes P077 BRCA2: Results obtained with P045 or P090 can be confirmed with this probemix. P090 BRCA2: Identical to P045 BRCA2/CHEK2, but does not contain probes for CHEK2. P002/P087 BRCA1: Hereditary breast cancer, screening BRCA1. P239 BRCA1 region: Characterization of BRCA1 region deletions/duplications. P190 CHEK2: Breast cancer susceptibility, genes included: CHEK2, ATM, BRCA1&2, PTEN, TP53. P057 FANCD2/PALB2: Mutations in PALB2 have been linked to a higher risk of breast cancer. P240 BRIP1/CHEK1: Mutations in BRIP1 have been linked to a higher risk of breast cancer. P041/P042 ATM: Mutations in ATM have been linked to a higher risk of breast cancer. References of SALSA probemix P045 Easton (1999). How many more breast cancer predisposition genes are there? Breast Cancer Res 1:14. Fachal et al. (2014). Large Genomic Rearrangements of BRCA1 and BRCA2 among Patients Referred for Genetic Analysis in Galicia (NW Spain): Delimitation and Mechanism of Three Novel BRCA1 Rearrangements. PLoS One 9:e93306. Janavičius et al. (2014). Comprehensive BRCA1 and BRCA2 mutational profile in Lithuania. Cancer Genet 207:195. Pal et al. (2005). BRCA1 and BRCA2 mutations account for a large proportion of ovarian carcinoma cases. Cancer 104:2807. Seong et al. (2014). A multi-institutional study of the prevalence of BRCA1 and BRCA2 large genomic rearrangements in familial breast cancer patients. BMC Cancer 14:645. Silva et al. (2014). Hereditary breast and ovarian cancer: assessment of point mutations and copy number variations in Brazilian patients. BMC Med Genet 15:55. Tedaldi et al. (2014). First evidence of a large CHEK2 duplication involved in cancer predisposition in an Italian family with hereditary breast cancer. BMC Cancer 14:478. Data analysis This P045-B3 BRCA2/CHEK2 probemix contains 44 different MLPA probes with amplification products between 130 and 495 nt. Please note that the 495 nt probe is specific for the CHEK2 1100delC mutation and will only appear in samples containing this mutation. In addition, the P045-B3 BRCA2/CHEK2 probemix contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA denaturation control fragments (D-fragments) at 88-92-96 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 www.mlpa.com. SALSA P045 BRCA2/CHEK2 probemix Page 2 of 8
SD029 Sample DNA The SD029 Sample DNA provided with this probemix can be used as Binning DNA sample for binning of mutation-specific probe CHEK2 probe 01772-L15680 1100delC. Inclusion of one reaction with 5 µl SD029 DNA in MLPA experiments is recommended as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software and as an artificial positive control for the specific point mutation. Binning SD 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 to use DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For further details, please consult the SD029 Binning DNA product description provided (also available online: www.mlpa.com). This product is for research use only (RUO). CHEK2 1100delC probe We have received reports of experiments at three different laboratories in which the CHEK2 1100delC peak spuriously appeared in known normal controls as well as all samples. At we have noticed this same phenomenon once with a similar CHEK2 probe in the P056 TP53 probemix. This did not seem to be a contamination problem, as it appeared in some experiments but not in others in which the same vials of probemix and reagents were used. Despite many attempts with variations in the protocol, we have not been able to reproduce this. Results obtained with this CHEK2 mutation probe should therefore be treated with caution. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P045 BRCA2/CHEK2 probemix Page 3 of 8
Table 1. SALSA MLPA P045-B3 BRCA2/CHEK2 probemix Length Chromosomal position SALSA MLPA probe (nt) reference CHEK2 BRCA2 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 88-92-96 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 00797-L00463 5q31 137 BRCA2 probe 02283-L12281 Exon 1 148 BRCA2 probe 02285-L01776 Exon 1 154 BRCA2 probe 09297-L08066 Exon 14 160 FRY probe 02143-L09586 20 kb before BRCA2 166 BRCA2 probe 02486-L01985 Exon 2 172 + BRCA2 probe 08898-L09587 Exon 3 178 BRCA2 probe 01599-L10642 Exon 3 184 Reference probe 01217-L00694 4q35 191 BRCA2 probe 09812-L10643 Exon 23 197 BRCA2 probe 01600-L04671 Exon 4 202 BRCA2 probe 08265-L08128 Exon 7 211 Reference probe 02333-L01826 12q23 220 BRCA2 probe 01602-L01184 Exon 8 229 BRCA2 probe 01603-L01185 Exon 9 238 Reference probe 00517-L00097 2q13 247 BRCA2 probe 01604-L01186 Exon 10 256 BRCA2 probe 02279-L01770 Exon 11 start 265 HSCB (HSC20) probe 06800-L02040 Promoter region 274 BRCA2 probe 01606-L01188 Exon 11 end 283 BRCA2 probe 01607-L01189 Exon 12 292 Reference probe 03018-L02458 12q13 301 BRCA2 probe 02280-L01771 Exon 13 309 BRCA2 probe 09809-L10257 Exon 5 319 BRCA2 probe 09296-L11090 Exon 27 326 BRCA2 probe 01610-L01192 Exon 15 337 BRCA2 probe 01611-L01193 Exon 16 346 BRCA2 probe 04585-L03983 Exon 6 355 BRCA2 probe 02281-L01772 Exon 17 364 BRCA2 probe 01613-L01195 Exon 18 373 Reference probe 02667-L04984 11q22 382 BRCA2 probe 01614-L01196 Exon 19 391 BRCA2 probe 08266-L08129 Exon 20 400 CHEK2 probe 02579-L12282 Exon 9 (10) 409 BRCA2 probe 02069-L01970 Exon 21 418 BRCA2 probe 01617-L01199 Exon 22 427 Reference probe 06942-L06522 11q12 436 BRCA2 probe 08267-L08130 Exon 24 445 BRCA2 probe 08268-L08131 Exon 25 454 N4BP2L1 (CG018) probe 02144-L01619 8 kb after BRCA2 463 BRCA2 probe 11984-L15346 Exon 26 476 BRCA2 probe 09293-L15678 Exon 27 486 Reference probe 05028-L15679 2q32 495 CHEK2 probe 01772-L15680 Exon 11 (12) Important information on this probe can be found below Table 2. + Warning: SNP rs115376548 can influence the signal of the 172 nt probe. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. Note: The CHEK2 exon numbering has changed. We now use the exon numbering according to the NCBI NM_007194.3 reference sequence. Between brackets the exon numbering used in version 1-19 of the product description. SALSA P045 BRCA2/CHEK2 probemix Page 4 of 8
Table 2. P045 probes arranged according to chromosomal location Table 2a. BRCA2 Length (nt) SALSA MLPA probe BRCA2 Exon Ligation site NM_000059.3 Partial sequence (24 nt adjacent to ligation site) Distance to next probe 160 02143-L09586 FRY gene GGCCCAGAGTTA-CCGAGTCCTCAC 20.1 kb start codon 228-230 (ex 2) 137 02283-L12281 Exon 1 0-1 CAGCGCGGGCTT-GTGGCGCGAGCT 0.2 kb 148 02285-L01776 Exon 1 22 nt after exon 1 TGGTAGTGGGTT-GGGACGAGCGCG 0.8 kb 166 02486-L01985 Exon 2 271-270; reverse AGCGTGTCTTAA-AAATTTCAAAAA 2.8 kb 178 01599-L10642 Exon 3 472-473 AATAATATTCAA-AGAGCAAGGGCT 0.2 kb 172 + 08898-L09587 Exon 3 107 nt after exon 3 CTGGGCAAATCA-GTCTCTCTGGCC 5.7 kb 197 01600-L04671 Exon 4 569-570 AATAGTAGACAT-AAAAGTCTTCGC 1.0 kb 309 09809-L10257 Exon 5 688-689 TGTAACACCACA-AAGAGATAAGTC 0.1 kb 346 04585-L03983 Exon 6 728-727; reverse ACAAACTTTGGT-GTATGAAACAAA 0.3 kb 202 08265-L08128 Exon 7 812-813 ATGTCTTGGTCA-AGTTCTTTAGCT 2.6 kb 220 01602-L01184 Exon 8 264 nt before exon 8 TCTGACTTTCCA-ACTCATTGTGGA 1.8 kb 229 01603-L01185 Exon 9 1001-1002 AACACAAATCAA-AGAGAAGCTGCA 1.6 kb 247 01604-L01186 Exon 10 1374-1375 GAAGTGACAAAA-TCTCCAAGGAAG 3.7 kb 256 02279-L01770 Exon 11 2192-2193 TCTGAAGAACCA-ACTTTGTCCTTA 4.8 kb 274 01606-L01188 Exon 11 6992-6993 TCTCTTTTTACA-TGTCCCGAAAAT 3.3 kb 283 01607-L01189 Exon 12 183 nt before exon 12 AAACAGAACAAA-AATGTAATTGAC 2.5 kb 301 02280-L01771 Exon 13 7216-7215; reverse GTACACAGGTAA-TCGGCTCTAAAG 8.2 kb 154 09297-L08066 Exon 14 7394-7395 TCTGCTACAAGA-AATGAAAAAATG 1.5 kb 326 01610-L01192 Exon 15 7762-7763 CAGTCTGTATCT-TGCAAAAACATC 1.3 kb 337 01611-L01193 Exon 16 7975-7976 ACAGTTGGCTGA-TGGTGGATGGCT 4.8 kb 355 02281-L01772 Exon 17 8158-8157; reverse TTAGGCATCTAT-TAGCAAATTCCT 0.8 kb 364 ~ 01613-L01195 Exon 18 8482-8483 TCAGAAGATTAT-TCTTCATGGAGC 7.0 kb 382 01614-L01196 Exon 19 8602-8603 GTATACCAAACT-TGGATTCTTTCC 0.5 kb 391 08266-L08129 Exon 20 8743-8744 ATCTGGATTATA-CATATTTCGCAA 5.7 kb 409 02069-L01970 Exon 21 8909-8910 ACAAGACAGCAA-GTTCGTGCTTTG 2.7 kb 418 01617-L01199 Exon 22 9100-9101 TGCTGAACAAAA-GGAACAAGGTTT 0.3 kb 191 09812-L10643 Exon 23 9214-9215 ATCATCAGATTT-ATATTCTCTGTT 0.3 kb 436 08267-L08130 Exon 24 9455-9454; reverse GAAACGACAAAT-CCTATTAGGTCC 14.8 kb 445 08268-L08131 Exon 25 9706-9707 AGAGACATTCAA-CAAAATGAAAAA 2.0 kb 463 11984-L15346 Exon 26 9786-9787 TACTGCATGCAA-ATGATCCCAAGT 1.3 kb 476 ± 09293-L15678 Exon 27 9988-9989 AAAGTCTTGTAA-AGGGGAGAAAGA 0.6 kb 319 ± 09296-L11090 Exon 27 10638-10639 ATCGGGCAAAAA-TCGTTTTGCCCG 8.4 kb stop codon 10482-10484 (ex 27) N4BP2L1 454 02144-L01619 CATTATTATTGA-TAATACCAACCT (CG018) gene The NM_000059.3 sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. CHEK2 Length (nt) SALSA MLPA probe CHEK2 Exon Ligation site NM_007194.3 Partial sequence (24 nt adjacent to ligation site) Distance to next probe 265 06800-L02040 HSCB gene/ CHEK2 2.1 kb before exon 1 TGGCTGAAGAAA-TCTGGGTGGACA 44.0 kb promoter 400 ^ 02579-L12282 Exon 9 (10) 1008-1009 CTGTTTGACAAA-GTGGTGGGGAAT 4.0 kb 495 ^ 01772-L15680 Exon 11 (12) 1173-1171; reverse TGCCCAAAATCA-TAATCTAAAATT The NM_007194.3 sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. SALSA P045 BRCA2/CHEK2 probemix Page 5 of 8
The 172 nt exon 3 probe will give a 50% reduced probe signal when the exon 3, 504del 5068insCCAT mutation is present (http://cancerres.aacrjournals.org/content/58/7/1372.abstract). The 178 nt exon 3 probe is not influenced by this deletion which has a breakpoint within BRCA2 exon 3. + Warning: SNP rs115376548 can influence the signal of the 172 nt probe. In case of apparent deletions, it is recommended to sequence the target site. A copy number change of the more variable exon 5 probe is unlikely in case the exon 6 probe, which is at very close distance, has no copy number change. The benign BRCA2 polymorphism 2192C>G (P655R) may reduce the signal of the 256 nt exon 11 probe (false positive signal). ~ We have been informed by lab in Oslo of a sequence variation in the region targeted by this probe (01613-L01195). The variant BRCA2 c.8229_8243del15 results in frame deletion of 5 AA (p.arg2744_gly2748del). The pathogenicity of this variant is not yet clear. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. ± The two exon 27 probes have a higher standard deviation than average. Apparent deletion of only one exon 27 probe could be a false positive. ^ The CHEK2 exon numbering has changed. We now use the exon numbering according to the NCBI NM_007194.3 reference sequence. Between brackets the exon numbering in versions 1-19 of the product description. Mutation 1100delC-specific! This peak will only appear if the mutation is present. The current 1100delC mutation specific probe cannot distinguish between the 1100delC sequence change present in the CHEK2 gene or its pseudogene. In case a positive signal is obtained with this probe, Sanger sequencing can clarify whether the mutation is present in the actual gene. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. Note: Exon numbering used here may differ from literature! Complete probe sequences and the identity of the genes detected by the reference probes are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P045 BRCA2/CHEK2 probemix Page 6 of 8
SALSA MLPA probemix P045-B3 BRCA2/CHEK2 sample pictures Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P045-B3 BRCA2/CHEK2 (lot B3-0715). Figure 2. Capillary electrophoresis pattern of SD029 Sample DNA (approximately 50 ng) analysed with SALSA MLPA probemix P045-B3 BRCA2/CHEK2 (lot B3-0715). The location of the mutation-specific probe at 495 nt is indicated. SALSA P045 BRCA2/CHEK2 probemix Page 7 of 8
Implemented Changes compared to the previous product description version(s). Version 32 (55) 09 September 2015 - Product description adapted to a new lot (lot number added, new pictures included). - Manufacturer s address adjusted. Version 32 (54) 27 May 2015 - Information about the BRCA2 added in the first paragraph and references updated. Version 31 (54) - 18 February 2015 - Exon numbering of the CHEK2 gene changed in Table 1 and Table 2b. Version 30 (54) - 30 January 2015 - Information about 1100delC mutation specific probe is added. - Information about Binning DNA SD029 added on page 1 and 2. - Change in format of date in header. - Change in text below figure 2. Version 29 (53) - Product description adapted to a new lot (lot number added and new picture included). - References for P045 probemix adjusted. Version 28 (53) - Remark added about 265nt probe: flanking probe. Version 27 (53) - Product description adapted to a new lot (lot number added and new picture included). Version 26 (52) - Warning about salt sensitivity of 400 nt probe removed. Version 25 (49) - Textual change below Table 2. Version 24 (49) - Product description adapted to a new lot (lot number added and new picture included). Version 23 (48) - Warning added in Table 1 and 2, 172 nt probe 08898-L09587. Version 22 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 21 (48) - Grammatical correction on page 1 and 3. Version 20 (48) - CHEK2 exon numbering changed according to reference standard in the RefSeqGene project. - Various textual changes in Tables 1 and 2. Version 19 (48) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new pictures included). Version 18 (48) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new pictures included). - CHEK2 exon numbering changed to the NCBI exon numbering. - Information about reference sequence added below Table 2. - Warning about 400 nt probe 02579-L12282 added to Table 1. - Warning about SNP in 172 nt probe target site added to Table 2. - Partial sequences extended to 24 nt. Version 17 (46) - Warning added for a sequence variant under table 1 and 2 for probe (01613-L01195) at 364 nt. - Tables have been numbered. - Small layout changes under table 1 on page 3. Version 16 (46) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new pictures included). - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. SALSA P045 BRCA2/CHEK2 probemix Page 8 of 8