MRC-Holland MLPA. Description version 18; 09 September 2015

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1 SALSA MLPA probemix P090-A4 BRCA2 Lot A4-0715, A4-0714, A4-0314, A4-0813, A4-0712: Compared to lot A3-0710, the 88 and 96 nt control fragments have been replaced (QDX2). This product is identical to the P045 BRCA2-CHEK2 probemix, but does not contain the three CHEK2 probes. This product is not intended for confirmation of results obtained with the P045 probemix. As the increased risk of breast cancer due to CHEK2 deletions is not well known, we received several requests to develop a product similar to P045 but without CHEK2 probes. Confirmation of P045/P090 results can be done with the P077 probemix. BREAST CARCINOMA is the most common malignancy among women in developed countries and family history remains the strongest single predictor of breast cancer risk. Mutations in the BRCA1 and BRCA2 genes are linked to a high risk of young-onset breast cancer, and appear to be responsible for approximately 10% of total breast cancer cases. Women with these mutations have a cumulative risk of developing breast cancer (up to age 70) of 55-85%. The BRCA1 and BRCA2 proteins are associated with the activation of double-strand break repair and/or homologous recombination. Unlike BRCA1, BRCA2 has not been linked to ovarian cancer. BRCA2 mutations are less frequent than BRCA1 mutations but in families with male breast cancer cases, BRCA2 mutations may be more frequent. Mutations in BRCA2 have been linked to several other types of cancers including ovarian, prostate and pancreatic cancer. The P090-A4 probemix contains probes for all exons of the BRCA2 gene. Two probes are present for exons 1, 3, 11 and 27. In addition, two probes are present detecting sequences just before and after the BRCA2 gene. Finally, 10 reference probes are included, detecting different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the BRCA2 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. 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 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 MLPA probemixes P077 BRCA2: Results obtained with P045/P090 can be confirmed with this probemix. P045 BRCA2/CHEK2: BRCA2 probes identical to P090, but also contains CHEK2 probes. P002/P087 BRCA1: Hereditary breast cancer, screening BRCA1. P239 BRCA1 region: Characterization of deletions/duplications upstream and downstream of BRCA1. 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 of breast cancer. P240 BRIP1/CHEK1: Mutations in BRIP1 has been linked to a higher risk of breast cancer. P041/P042 ATM: Mutations in ATM has been linked to a higher risk of breast cancer. More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA P090 BRCA2 probemix Page 1 of 6

2 Selection of references of SALSA MLPA probemix P045/P090 Akbari MR, et al. (2014). The spectrum of BRCA1 and BRCA2 mutations in breast cancer patients in the Bahamas. Clin Genet. 85:64-7. Fachal L, 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:e Rudnicka H, et al. (2014). Large BRCA1 and BRCA2 genomic rearrangements in Polish high-risk breast and ovarian cancer families. Mol Biol Rep. 40: Peixoto A, et al. (2013). Genomic characterization of two large Alu-mediated rearrangements of the BRCA1 gene. J Hum Genet. 58: Ruiz de Garibay G, et al. (2012). Characterization of four novel BRCA2 large genomic rearrangements in Spanish breast/ovarian cancer families: review of the literature, and reevaluation of the genetic mechanisms involved in their origin. Breast Cancer Res Treat. 133: Kuusisto KM, et al. (2011). Screening for BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 mutations in high-risk Finnish BRCA1/2-founder mutation-negative breast and/or ovarian cancer individuals. Breast Cancer Res. 13:R20. Del Valle J, et al. (2009). Identification and comprehensive characterization of large genomic rearrangements in the BRCA1 and BRCA2 genes. Breast Cancer Res Treat. 122: Hansen TO, et al. (2009). Large BRCA1 and BRCA2 genomic rearrangements in Danish high risk breastovarian cancer families. Breast Cancer Res Treat. 115: Data analysis The P090-A4 BRCA2 probemix contains 43 MLPA probes with amplification products between 130 and 486 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 (D-fragments) 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 Many copy number alterations in healthy individuals are described in the database of genomic variants: For example, a duplication of a complete gene might not be pathogenic, while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions may result in a very mild, or no disease. Copy number changes of reference probes are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P090 BRCA2 probemix Page 2 of 6

3 Table 1. SALSA MLPA P090-A4 BRCA2 probemix Length Chromosomal position SALSA MLPA probe (nt) 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 130 Reference probe L q BRCA2 probe L12281 Exon BRCA2 probe L13497 Exon BRCA2 probe L08066 Exon FRY probe L kb upstream BRCA2 166 BRCA2 probe L01985 Exon BRCA2 probe L09587 Exon BRCA2 probe L10642 Exon Reference probe L q BRCA2 probe L10643 Exon BRCA2 probe L04671 Exon BRCA2 probe L08128 Exon Reference probe L q BRCA2 probe L01184 Exon BRCA2 probe L01185 Exon Reference probe L q BRCA2 probe L01186 Exon BRCA2 probe L01770 Exon Reference probe L p BRCA2 probe L01188 Exon BRCA2 probe L01189 Exon Reference probe L q BRCA2 probe L01771 Exon BRCA2 probe L10257 Exon BRCA2 probe L11090 Exon BRCA2 probe L01192 Exon BRCA2 probe L01193 Exon BRCA2 probe L03983 Exon BRCA2 probe L01772 Exon BRCA2 probe L01195 Exon Reference probe L q BRCA2 probe L01196 Exon BRCA2 probe L08129 Exon Reference probe L q BRCA2 probe L01970 Exon BRCA2 probe L01199 Exon Reference probe L q BRCA2 probe L08130 Exon BRCA2 probe L08131 Exon N4BP2L1 (=CG018) probe L kb downstream BRCA2 463 BRCA2 probe L15346 Exon BRCA2 probe L15678 Exon Reference probe L q32 Important information on this probe can be found in and below Table 2. Warning: We have been informed that the signal of the 172 nt probe, L09587 may be hampered by the presence of a SNP (0.05*) rs SALSA P090 BRCA2 probemix Page 3 of 6

4 Table 2. P090 BRCA2 probes arranged to chromosomal location Length (nt) SALSA MLPA probe BRCA2 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe L09586 FRY gene GGCCCAGAGTTA-CCGAGTCCTCAC 20.1 kb Start codon (ex 2) L12281 Exon CAGCGCGGGCTT-GTGGCGCGAGCT 0.2 kb L13497 Exon 1 22 nt after exon 1 TGGTAGTGGGTT-GGGACGAGCGCG 0.8 kb L01985 Exon reverse AGCGTGTCTTAA-AAATTTCAAAAA 2.8 kb L10642 Exon AATAATATTCAA-AGAGCAAGGGCT 0.2 kb L09587 Exon nt after exon 3 CTGGGCAAATCA-GTCTCTCTGGCC 5.7 kb L04671 Exon AATAGTAGACAT-AAAAGTCTTCGC 1.0 kb L10257 Exon TGTAACACCACA-AAGAGATAAGTC 0.1 kb L03983 Exon reverse ACAAACTTTGGT-GTATGAAACAAA 0.3 kb L08128 Exon ATGTCTTGGTCA-AGTTCTTTAGCT 2.6 kb L01184 Exon nt before exon 8 TCTGACTTTCCA-ACTCATTGTGGA 1.8 kb L01185 Exon AACACAAATCAA-AGAGAAGCTGCA 1.6 kb L01186 Exon GAAGTGACAAAA-TCTCCAAGGAAG 3.7 kb 256 ± L01770 Exon TCTGAAGAACCA-ACTTTGTCCTTA 4.8 kb L01188 Exon TCTCTTTTTACA-TGTCCCGAAAAT 3.3 kb L01189 Exon nt before exon 12 AAACAGAACAAA-AATGTAATTGAC 2.5 kb L01771 Exon reverse GTACACAGGTAA-TCGGCTCTAAAG 8.2 kb L08066 Exon TCTGCTACAAGA-AATGAAAAAATG 1.5 kb L01192 Exon CAGTCTGTATCT-TGCAAAAACATC 1.3 kb L01193 Exon ACAGTTGGCTGA-TGGTGGATGGCT 4.8 kb L01772 Exon reverse TTAGGCATCTAT-TAGCAAATTCCT 0.8 kb 364 ~ L01195 Exon TCAGAAGATTAT-TCTTCATGGAGC 7.0 kb L01196 Exon GTATACCAAACT-TGGATTCTTTCC 0.5 kb L08129 Exon ATCTGGATTATA-CATATTTCGCAA 5.7 kb L01970 Exon ACAAGACAGCAA-GTTCGTGCTTTG 2.7 kb L01199 Exon TGCTGAACAAAA-GGAACAAGGTTT 0.3 kb L10643 Exon ATCATCAGATTT-ATATTCTCTGTT 0.3 kb L08130 Exon reverse GAAACGACAAAT-CCTATTAGGTCC 14.8 kb L08131 Exon AGAGACATTCAA-CAAAATGAAAAA 2.0 kb L15346 Exon TACTGCATGCAA-ATGATCCCAAGT 1.3 kb L15678 Exon AAAGTCTTGTAA-AGGGGAGAAAGA 0.6 kb L11090 Exon ATCGGGCAAAAA-TCGTTTTGCCCG 8.4 kb Stop codon (ex 27) L01619 N4BP2L1 (CG018) gene CATTATTATTGA-TAATACCAACCT The 172 nt exon 3 probe will give a 50% reduced probe signal when the exon 3, nt504del5068insccat mutation is present ( The 178 nt exon 3 probe is not influenced by this deletion. Warning: We have been informed that the signal of the 172 nt probe, L09587 may be hampered by the presence of a SNP (0.05*) rs 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), can result in a lower probe signal of the 256 nt exon 11 probe (false positive signal). The two exon 27 probes each have a higher than average standard deviation. Several other exon 27 probes prepared at have also been somewhat variable in results. Apparent deletions of only one of the exon 27 probes only could well be a false positive. ~ We have been informed 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. 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. Notes: the NM_ sequence is a reference standard in the NCBI RefSeqGene project. Exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P090 BRCA2 probemix Page 4 of 6

5 SALSA MLPA probemix P090-A4 BRCA2 sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P090-A4 BRCA2 (lot A4-0715). SALSA P090 BRCA2 probemix Page 5 of 6

6 Implemented Changes compared to the previous product description version(s). Version 18 (55) 09 September Manufacturer s address adjusted. Version 17 (53) Version 16 (53) Version 15 (52) Version 14 (48) - Textual change below Table 2. Version 13 (48) - Warning added in Table 1 and 2, 172 nt probe L Version 12 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 11 (48) - 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). - Various minor textual changes. Version 10 (48) - Ligation sites of the probes targeting the BRCA2 gene updated according to new version of the NM_reference sequence. - Various minor textual changes. - Remark on RefSeqGene standard added below Table 2. Version 08 (46) - Product description adapted to a new lot and new product version (lot number added, small changes in Table 1 and Table 2, new picture included). - Section Many copy number alterations in healthy individuals has been added to page 2. Version 07 (46) - Warning added for a sequence variant under table 1 and 2 for probe (01613-L01195) at 364 nt. - Small layout changes under table 2 on page 4. Version 06 (45) - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - More references added, e.g. on the 504del 5068insCCAT mutation. SALSA P090 BRCA2 probemix Page 6 of 6