MRC-Holland MLPA. Description version 33; 09 September 2015

Transcription

1 SALSA MLPA probemix P045-B3 BRCA2/CHEK2 Lot B3-0715, B3-0714, B3-1113, B3-0413, lot B 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 : 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

2 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:e 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 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 SALSA P045 BRCA2/CHEK2 probemix Page 2 of 8

3 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 L delC. 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: 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

4 Table 1. SALSA MLPA P045-B3 BRCA2/CHEK2 probemix Length Chromosomal position SALSA MLPA probe (nt) reference CHEK2 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 L01776 Exon BRCA2 probe L08066 Exon FRY probe L kb before 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 11 start 265 HSCB (HSC20) probe L02040 Promoter region 274 BRCA2 probe L01188 Exon 11 end 283 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 CHEK2 probe L12282 Exon 9 (10) 409 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 after BRCA2 463 BRCA2 probe L15346 Exon BRCA2 probe L15678 Exon Reference probe L q CHEK2 probe L15680 Exon 11 (12) Important information on this probe can be found below Table 2. + Warning: SNP rs 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_ 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

5 Table 2. P045 probes arranged according to chromosomal location Table 2a. BRCA2 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 L01776 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 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 476 ± L15678 Exon AAAGTCTTGTAA-AGGGGAGAAAGA 0.6 kb 319 ± L11090 Exon ATCGGGCAAAAA-TCGTTTTGCCCG 8.4 kb stop codon (ex 27) N4BP2L L01619 CATTATTATTGA-TAATACCAACCT (CG018) gene The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. CHEK2 Length (nt) SALSA MLPA probe CHEK2 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe L02040 HSCB gene/ CHEK2 2.1 kb before exon 1 TGGCTGAAGAAA-TCTGGGTGGACA 44.0 kb promoter 400 ^ L12282 Exon 9 (10) CTGTTTGACAAA-GTGGTGGGGAAT 4.0 kb 495 ^ L15680 Exon 11 (12) ; reverse TGCCCAAAATCA-TAATCTAAAATT The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. SALSA P045 BRCA2/CHEK2 probemix Page 5 of 8

6 The 172 nt exon 3 probe will give a 50% reduced probe signal when the exon 3, 504del 5068insCCAT mutation is present ( The 178 nt exon 3 probe is not influenced by this deletion which has a breakpoint within BRCA2 exon 3. + Warning: SNP rs 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_ 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

7 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

8 Implemented Changes compared to the previous product description version(s). Version 32 (55) 09 September Product description adapted to a new lot (lot number added, new pictures included). - Manufacturer s address adjusted. Version 32 (54) 27 May Information about the BRCA2 added in the first paragraph and references updated. Version 31 (54) - 18 February Exon numbering of the CHEK2 gene changed in Table 1 and Table 2b. Version 30 (54) - 30 January 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 L 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 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