SALSA MLPA probemix P323-B1 CDK4-HMGA2-MDM2 Lot B1-0711. As compared to version A1 (test version sent to test labs), this product has been completely redesigned. Probes for HMGA2 and several other genes at 12p and 12q have been included. In addition, the 88 and 96 nt control fragments have been replaced (QDX2). Copy number changes of chromosome 12 and amplified segments of 12q arm are frequently observed in several types of sarcomas and also in other tumours. These amplifications can be used in defining genetically distinct subsets of tumours with differential diagnosis and/or prognosis. The P323 CDK4-HMGA-MDM2 probemix can be used to detect copy number changes of chromosome 12 and to detect amplifications of the MDM2, CDK4 and HMGA2 genes. Please note that this SALSA probemix is intended for research purposes and has not been validated yet on a sufficient number of patient samples. In liposarcomas, MDM2 and HMGA2 genes are consistently amplified in well-differentiated (WDLPS) and dedifferentiated (DDLPS) types of liposarcomas, which can be used clinically in distinguishing them from benign lipomas (Italiano A. et al. 2008. Int J. Cancer). Moreover, DDLPS and WDLPS patients with only MDM2-HMGA2 amplification have a favourable prognosis compared to patients with both HMGA2-MDM2 and CDK4 amplifications (Italiano A. et al. 2009, Clin Cancer Res). In osteosarcoma (OS), MDM2-CDK4 amplification can be used in differential diagnostics, since MDM2-CDK4 amplification seems to be most prevalent in parosteal OS (Mejia-Guerrero S. et al. 2010, Genes Chromosomes Cancer). Amplifications of 12q13-14 (including CDK4, HMGA2, MDM2, TSPAN31 and GLI1 genes) are common in leiomyosarcoma and alveaolar/embryonal/sclerosing rhabdomyosarcoma and 12q13-q14 amplifications in alveolar rhabdomyosarcoma correlate with poor survival (Barr F. et al. 2009, Genes Chromosomes Cancer). HMGA2 amplifications are characteristic for pituitary adenomas, and especially to prolactiomas (Finelli P. et al. 2002, Cancer Res). HMGA2 amplifications are also observed in adenomas and carcinomas of salivary glands (Persson F. et al. 2009, Genes Chromosomes Cancer). This probemix contains 34 probes detecting copy number changes of chromosome 12, including 2 probes for CDK4, 5 probes for HMGA2 and 4 probes for the MDM2 gene. In addition, 12 reference probes are included in this probe mix detecting 12 different autosomal chromosome locations, which are relatively quiet in sarcomas and tumours in general. However, it should be noticed, that tumour karyotypes can harbour multiple numerical and structural aberrations, which can complicate interpretation of these reference probes. This SALSA probemix is designed to detect copy number changes of one or more sequences in the above mentioned genes and chromosomal regions 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, apparent deletions detected by a single probe always require confirmation by other methods. Moreover, users should always verify the latest scientific literature when interpreting their findings. SALSA probemixes are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. This probemix is not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA MLPA test probemixes includes a limited license to use these products for research purposes. The use of this SALSA probemix 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 6, 1057 DN Amsterdam, the Netherlands SALSA probemix P323 CDK4-HMGA2-MDM2 Page 1 of 5
Data analysis The P323-B1 CDK4-HMGA-MDM2 probemix contains 46 MLPA probes with amplification products between 122 and 456 nt. In addition, it 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 should be normalised with a robust method, as the target sites of the reference probes maybe gained or lost. (1) Intra-sample normalisation should be performed by dividing the signal of each target-specific probe by the signal of every single reference probe in that sample, thus creating as many ratios per target-specific probe as there are reference probes. Subsequently, the median of all these produced ratios per probe should be taken; this is the probe s Normalisation Constant. (2) Secondly, inter-sample comparison should be performed by dividing the Normalisation Constant of each probe in a given sample by the average Normalisation Constant of that probe in all the reference samples. Data normalisation should be performed within one experiment. Always use sample and reference DNA extracted with the same method and derived from the same source of tissue. Confirmation of deletions, duplications 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. MLPA analysis on tumour samples provides information on the average situation in the cells from which the DNA sample was purified. Gains or losses of genomic regions or genes may not be detected if the percentage of tumour cells is low. Reference probes are located in silent regions that are not frequently altered in copy number in liposarcoma, osteosarcome, rhabdomyosarcoma, adenomas/carcinomas of salivary gland and pituitary adenomas, but there is always a possibility that one or more reference probes have a copy number alteration in a sample. This probemix was developed by H. Yigittop and S. Savola at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designers could be made co-authors. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P323 CDK4-HMGA2-MDM2 Page 2 of 5
Table 1. SALSA MLPA P323-B1 MDM2-CDK4 probemix Length Chromosomal position SALSA MLPA probe (nt) Reference 12p 12q 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 86-92-96 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 122 Reference probe 02844-L02274 18q11 130 Reference probe 00797-L13645 5q31 136 MAP3K12 probe 15901-L17994 12q13.13 142± CDK4 probe 03173-L02512 12q14.1 148 HMGA2 probe 15075-L16838 12q14.3 154 GLI1 probe 15902-L17995 12q13.3 160 TBX5 probe 05694-L05136 12q24.21 166 Reference probe 14281-L15951 15q13 172 CCND2 probe 03177-L02516 12p13.32 178 Reference probe 04446-L04183 4q13 184 KRAS probe 10517-L11071 12p12.1 190 MDM2 probe 07182-L06791 12q15 196 Reference probe 05300-L04688 3q11 202 TSPAN31 probe 15903-L18385 12q14.1 208 ALX1 probe 14414-L16627 12q21.31 216 Reference probe 14640-L18386 19q13 226 PIWIL1 probe 09841-L18685 12q24.33 232 FOXM1 probe 07325-L18686 12p13.33 238 CHFR probe 02738-L18389 12q24.33 247 Reference probe 07239-L06889 3p11 253 HNF1A probe 07717-L18390 12q24.31 262 MDM2 probe 07183-L18784 12q15 267 CDK4 probe 15904-L18392 12q14.1 274 YEATS4 probe 15905-L18393 12q15 283 HMGA2 probe 16186-L16821 12q14.3 292 Reference probe 07017-L06628 14q11 300 RAN probe 15906-L18397 12q24.33 310 IGF1 probe 02340-L01834 12q23.2 317± CDKN1B probe 16517-L18978 12p13.1 324 MIR26A2 probe 16903-L20362 12q14.1 330 DDIT3 probe 15907-L20363 12q13.3 338 MDM2 probe 02894-L20364 12q15 346 GLI1 probe 15908-L18001 12q13.3 355 RAN probe 15909-L18002 12q24.33 362 KIF21A probe 05762-L18394 12q12 370 MDM2 probe 00337-L18786 12q15 377 Reference probe 06216-L20365 16p11 385 Reference probe 05914-L05359 18p11 392 CCND2 probe 03178-L18979 12p13.32 400 CDK2 probe 14405-L16087 12q13.2 409 PTPN11 probe 12523-L13573 12q24.1 418 ± HMGA2 probe 15074-L16832 12q14.3 427 HMGA2 probe 15084-L16847 12q14.3 436 Reference probe 05504-L04927 10q11 445 HMGA2 probe 15086-L16849 12q14.3 456 Reference probe 13470-L20366 2q13 ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Homology between Lig and reverse primer sequence results in high primer dimers when using the old primer mix and old 2 tube protocol and the PCR is started at room temperature. PCR then requires hot start. No problems are expected with new primer mix and 1 tube protocol. Note: Exon numbering used here may differ from literature! 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 P323 CDK4-HMGA2-MDM2 Page 3 of 5
Table 2. P323 probes arranged according to chromosomal location Length SALSA MLPA Gene Partial sequence (24 nt Distance to MV location Location (nt) probe Exon adjacent to ligation site) next probe (HG18) Reference probes 456 13470-L20366 PAX8 2q13 TTGCAGATGCTA-GGACACAAGAGA 02-113.719 247 07239-L06889 POU1F1 3p11.2 TCCTATACACCA-GCCTCTTCTGGC 7691.4 kb 03-087.396 196 05300-L04688 PROS1 3q11.2 CATTTAAATCCC-CAGCATAAATCA 03-095.088 178 04446-L04183 GNRHR 4q13.2 TGGAACATTACA-GTCCAATGGTAT 04-068.302 130 00797-L13645 IL4 5q31.1 ATCGACACCTAT-TAATGGGTCTCA 05-132.038 436 05504-L04927 RET 10q11.2 CCTGCAACTGCT-TCCCTGAGGAGG 10-042.929 12p chromosomal arm 232 07325-L18686 FOXM1, ex 4 12p13.33 CCATGATACAAT-TCGCCATCAACA 1405.1 kb 12-002.848 172 03177-L02516 CCND2, ex 1 12p13.32 AGACCAGTTTTA-AGGGGAGGACCG 29.9 kb 12-004.253 392 03178-L18979 CCND2, ex 5 12p13.32 TAACAGCCAAGA-AGCCTGCAGGAG 8478.8 kb 12-004.283 317 16517-L18978 CDKN1B, ex1 12p13.1 CGCGCTCCTAGA-GCTCGGGCCGTG 12527.6 kb 12-012.762 184 10517-L11071 KRAS, ex 2 12p12.1 ATTTTGTGGACG-AATATGATCCAA 12685.1 kb 12-025.289 12q chromosomal arm 362 05762-L18394 KIF21A, ex 38 12q12 AGGCTCGCAATT-TGCAAGATGGTC 14192.6 kb 12-037.975 136 15901-L17994 MAP3K12, ex 2 (3) 12q13.13 GCATCCAGAGTT-CGAGCTGACGAG 2479.8 kb 12-052.167 400 14405-L16087 CDK2, ex 1 12q13.2 CATTGTTTCAAG-TTGGCCAAATTG 1498.0 kb 12-054.647 154 15902-L17995 GLI1, ex 4 12q13.3 ACTCGCGATGCA-CATCTCCAGGAG 4.8 kb 12-056.145 346 15908-L18001 GLI1, ex 11 12q13.3 GGACCAGCTACA-TCAACTCCGGCC 47.1 kb 12-056.150 330 15907-L20363 DDIT3, ex 3 (4) 12q13.3 CCTCTCACTAGT-GCCAATGATGTG 229.1 kb 12-056.197 202 15903-L18385 TSPAN31, ex 2 12q14.1 TCCACATCATCG-GCGGAGTCATTG 2.8 kb 12-056.426 267 15904-L18392 CDK4, ex 8 12q14.1 TGCTGACTTTTA-ACCCACACAAGC 2.7 kb 12-056.429 142 03173-L02512 CDK4, ex 3 12q14.1 AACCCTGGTGTT-TGAGCATGTAGA 73.4 kb 12-056.431 324 16903-L20362 MIR26A2, ex 1 12q14.1 AGGCCTCACAGA-TGGAAACAGCCT 8000.2 kb 12-056.505 283 16186-L16821 HMGA2, ex 1 12q14.3 CCGCCTAACATT-TCAAGGGACACA 3.2 kb 12-064.505 418 15074-L16832 HMGA2, ex 2 12q14.3 GACCCAGGGGAA-GACCCAAAGGCA 10.5 kb 12-064.508 148 15075-L16838 HMGA2, ex 3 12q14.3 AGCCACTGGAGA-AAAACGGCCAAG 76.8 kb 12-064.519 445 15086-L16849 HMGA2, ex 4 12q14.3 CCAAGATGTAGT-TTCACTGCTACC 48.0 kb 12-064.595 427 15084-L16847 HMGA2, ex 6 12q14.3 AGTGACCACTTA-TTCTGTATTGCC 2844.9 kb 12-064.643 338 02894-L20364 MDM2, ex 1 12q15 CGAGATCCTGCT-GCTTTCGCAGCC 16.1 kb 12-067.488 370 00337-L18786 MDM2, ex 7 12q15 GTACATCTGTGA-GTGAGAACAGGT 0.2 kb 12-067.504 190 07182-L06791 MDM2, ex 8 12q15 GAGAAACCTTCA-TCTTCACATTTG 4.2 kb 12-067.505 262 07183-L18784 MDM2, ex 9 12q15 GAAAACGCCACA-AATCTGATAGTA 531.2 kb 12-067.509 274 15905-L18393 YEATS4, ex 1 12q15 TATGTTCAAGAG-AATGGCCGAATT 16158.3 kb 12-068.040 208 14414-L16627 ALX1, ex 1 12q21.31 GTCTGCAGGCAA-ATGCGTGCAGGC 17195.4 kb 12-084.198 310 02340-L01834 IGF1, ex 3 12q23.2 AGGTAGAAGAGA-TGCGAGGAGGAC 9947.3 kb 12-101.394 409 12523-L13573 PTPN11, ex 1 12q24.13 CAGGAGGAAGCA-AGGATGCTTTGG 1947.6 kb 12-111.341 160 05694-L05136 TBX5, ex 9 12q24.21 GTGAGGCAAAAA-GTGGCCTCCAAC 6633.1 kb 12-113.288 253 07717-L18390 HNF1A, ex 9 12q24.31 GCCTCAGTGTCT-GAGGTGAAGACC 9500.4 kb 12-119.922 226 09841-L18685 PIWIL1, ex 21 12q24.33 CAGAGAGCCAAA-TCTGTCACTGTC 501.3 kb 12-129.422 300 15906-L18397 RAN, ex 3 12q24.33 GTGTTTTTCAAC-AGCTTGTATTGG 1.7 kb 12-129.923 355 15909-L18002 RAN, ex 5 12q24.33 GTACTAATTCCC-ACAAATGTTTCT 2049.3 kb 12-129.925 238 02738-L18389 CHFR, ex 1 12q24.33 GGCGGCGGCGCT-CACCAAGAGCGG 2.5 Mb to 12q telomer 12-131.974 Reference probes 292 07017-L06628 RPGRIP1 14q11.2 CTACATCAGGAG-ACTTGCCAGTTA 14-020.826 166 14281-L15951 OCA2 15q13.1 GCCGCGATGAGA-CAGAGCATGATG 15-025.904 377 06216-L20365 TGFB1I1 16p11.2 CAGGAACTTAAT-GCCACTCAGTTC 16-031.393 385 05914-L05359 RNMT 18p11.21 TACAATGAACTT-CAGGAAGTTGGT 5669.9 kb 18-013.724 122 02844-L02274 NPC1 18q11.2 GACGAGTCTGTG-GATGAGGTCACA 18-019.394 216 14640-L18386 SDHAF1 19q13.12 AGCGTCTCTGCT-TAGCCGCGGTCA 19-041.178 Note: The DDIT3 and MAP3K12 exon numbering has changed. From description version 03 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for the DDIT3 and MAP3K12 genes. The exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. Complete probe sequences are available on request: info@mlpa.com. SALSA probemix P323 CDK4-HMGA2-MDM2 Page 4 of 5
SALSA MLPA probemix P323-B1 CDK4-HMGA2-MDM2 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P323-B1 CDK4-HMGA2-MDM2 (lot B1-0711). Implemented Changes compared to the previous product description version(s). Version 06 (48) 07 August 2015 - Electropherogram picture(s) using the old MLPA buffer (replaced in December 2012) removed. Version 05 (48) - Warning added in Table 1, 142 nt probe 03173-L02512 and 418 nt probe 15074-L16832. Version 04 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 03 (48) - Various minor textual changes on page 1. - Exon numbering of the DDIT3 and MAP3K12 genes have been changed in Table 2. - Small correction of chromosomal locations in Table 1 and 2. Version 02 (46) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). SALSA probemix P323 CDK4-HMGA2-MDM2 Page 5 of 5