SALSA MLPA probemix P323-B1 CDK4-HMGA2-MDM2 Lot B1-0714, B1-0711. As compared to previous test version (lot A1-0508), this probemix 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). Sarcomas represent a relatively rare group of cancers occurring in different types of tissues, such as fat, bones, muscles, nerves. Alteration of CDK4, MDM2 and HMGA2 genes are suggested to be diagnostically, clinically and prognostically relevant in liposarcoma, osteosarcoma, rhabdomyosarcoma, adenomas/ carcinomas of salivary gland and pituitary adenomas. In well-differentiated (WDLPS) and dedifferentiated (DDLPS) types of liposarcomas, MDM2 and HMGA2 genes are consistently amplified, which can differentiate those 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, as MDM2-CDK4 amplification seems to be most prevalent in parosteal OS (Mejia-Guerrero S. et al. 2010, Genes Chromosomes Cancer). Amplifications of 12q13-14 region (including CDK4, HMGA2, MDM2, TSPAN31 and GLI1 genes) are common in leiomyosarcoma and alveolar, embryonal and sclerosing rhabdomyosarcoma, and correlate with poor survival in alveolar rhabdomyosarcoma (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 P323-B1 CDK4-HMGA2-MDM2 probemix contains 34 probes for detecting copy number changes of chromosome 12, including two probes for CDK4, five probes for HMGA2 (one for each exon) and four probes for the MDM2 gene. In addition, 12 reference probes have been included in this probemix, detecting 12 different autosomal chromosomal locations which are relatively stable in sarcomas. However, it should be noticed that tumour karyotypes typically 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 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 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 P419 CDKN2A/2B-CDK4: Contains more probes for CDK4 gene, involved in sarcomas P175 Tumour Gain: Contains two other probes for MDM2 gene, involved in sarcomas References for SALSA P323-B1 CDK4-HMGA2-MDM2 probemix Fusco I et al. 2015. Variations in the high-mobility group-a2 gene (HMGA2) are associated with idiopathic short stature. Pediatr Res. PMID: 26536448. [Epub ahead of print] SALSA probemix P323 CDK4-HMGA2-MDM2 Page 1 of 6
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 Data analysis The P323-B1 CDK4-HMGA2-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 more 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. Warning: 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. Furthermore, although reference probes are located in silent regions that are not frequently altered in copy number in various sarcoma types, there is always a possibility that one or more reference probes do show a copy number alteration in a sample. Normal copy number variation in healthy individuals is described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. When in doubt, users should always verify the latest updates of the database and scientific literature when interpreting their findings. This probemix was developed by H. Yigittop at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designer could be included as co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P323 CDK4-HMGA2-MDM2 Page 2 of 6
Table 1. SALSA MLPA P323-B1 CDK4-HMGA2-MDM2 probemix Length Chromosomal position MV location SALSA MLPA probe (nt) reference 12p 12q (HG18) 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 122 Reference probe 02844-L02274 18q11.2 18-019.394343 130 Reference probe 00797-L13645 5q31.1 05-132.037613 136 MAP3K12 probe 15901-L17994 12q13.13 12-052.167097 142 «CDK4 probe 03173-L02512 12q14.1 12-056.431292 148 HMGA2 probe 15075-L16838 12q14.3 12-064.518559 154 GLI1 probe 15902-L17995 12q13.3 12-056.144842 160 ± TBX5 probe 05694-L05136 12q24.21 12-113.288495 166 Reference probe 14281-L15951 15q13.1 15-025.903847 172 CCND2 probe 03177-L02516 12p13.32 12-004.253202 178 Reference probe 04446-L04183 4q13.2 04-068.302296 184 KRAS probe 10517-L11071 12p12.1 12-025.289452 190 MDM2 probe 07182-L06791 12q15 12-067.504608 196 Reference probe 05300-L04688 3q11.1 03-095.087606 202 TSPAN31 probe 15903-L18385 12q14.1 12-056.425844 208 ALX1 probe 14414-L16627 12q21.31 12-084.198284 216 Reference probe 14640-L18386 19q13.12 19-041.177979 226 PIWIL1 probe 09841-L18685 12q24.33 12-129.422034 232 FOXM1 probe 07325-L18686 12p13.33 12-002.848060 238 CHFR probe 02738-L18389 12q24.33 12-131.974313 247 Reference probe 07239-L06889 3p11.2 03-087.396245 253 HNF1A probe 07717-L18390 12q24.31 12-119.921641 262 MDM2 probe 07183-L18784 12q15 12-067.508838 267 CDK4 probe 15904-L18392 12q14.1 12-056.428612 274 YEATS4 probe 15905-L18393 12q15 12-068.040001 283 HMGA2 probe 16186-L16821 12q14.3 12-064.504834 292 Reference probe 07017-L06628 14q11.2 14-020.825979 300 RAN probe 15906-L18397 12q24.33 12-129.923290 310 IGF1 probe 02340-L01834 12q23.2 12-101.393655 317 CDKN1B probe 16517-L18978 12p13.1 12-012.761863 324 MIR26A2 probe 16903-L20362 12q14.1 12-056.504665 330 DDIT3 probe 15907-L20363 12q13.3 12-056.196743 338 MDM2 probe 02894-L20364 12q15 12-067.488328 346 GLI1 probe 15908-L18001 12q13.3 12-056.149664 355 RAN probe 15909-L18002 12q24.33 12-129.924992 362 KIF21A probe 05762-L18394 12q12 12-037.974514 370 MDM2 probe 00337-L18786 12q15 12-067.504408 377 Reference probe 06216-L20365 16p11.2 16-031.392738 385 Reference probe 05914-L05359 18p11.21 18-013.724478 392 CCND2 probe 03178-L18979 12p13.32 12-004.283081 400 CDK2 probe 14405-L16087 12q13.2 12-054.646859 409 PTPN11 probe 12523-L13573 12q24.13 12-111.340923 418 «HMGA2 probe 15074-L16832 12q14.3 12-064.508064 427 HMGA2 probe 15084-L16847 12q14.3 12-064.643387 436 Reference probe 05504-L04927 10q11.21 10-042.929060 445 HMGA2 probe 15086-L16849 12q14.3 12-064.595338 456 Reference probe 13470-L20366 2q13 02-113.718910 «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. ± SNP rs147405081 and rs375955080 could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. The sequence targeted by this reference probe has been reported to be frequently affected by copy number changes in sarcoma samples. Data analysis should be performed with caution for this probe. SALSA probemix P323 CDK4-HMGA2-MDM2 Page 3 of 6
Table 2. P323 probes arranged according to chromosomal location Length SALSA MLPA Location/ Partial sequence Distance to Gene /Exon (nt) probe Ligation site (24 nt adjacent to ligation site) next probe Reference probes on chr. 2, 3, 4, 5 and 10. 456 13470-L20366 PAX8 2q13 TTGCAGATGCTA-GGACACAAGAGA 247 07239-L06889 POU1F1 3p11.2 TCCTATACACCA-GCCTCTTCTGGC 7691.4 kb 196 05300-L04688 PROS1 3q11.1 CATTTAAATCCC-CAGCATAAATCA 178 04446-L04183 GNRHR 4q13.2 TGGAACATTACA-GTCCAATGGTAT 130 00797-L13645 IL4 5q31.1 ATCGACACCTAT-TAATGGGTCTCA 436 05504-L04927 RET 10q11.21 CCTGCAACTGCT-TCCCTGAGGAGG 12p chromosomal arm 232 07325-L18686 FOXM1, ex 4 12p13.33 CCATGATACAAT-TCGCCATCAACA 1405.1 kb 172 03177-L02516 CCND2, ex 1 NM_001759.3; 68-69 AGACCAGTTTTA-AGGGGAGGACCG 29.9 kb 392 03178-L18979 CCND2, ex 5 NM_001759.3; 4844-4845 TAACAGCCAAGA-AGCCTGCAGGAG 8478.8 kb 317 16517-L18978 CDKN1B, ex 1 12p13.1 CGCGCTCCTAGA-GCTCGGGCCGTG 12527.6 kb 184 10517-L11071 KRAS, ex 2 12p12.1 ATTTTGTGGACG-AATATGATCCAA 12685.1 kb 12q chromosomal arm 362 05762-L18394 KIF21A, ex 38 12q12 AGGCTCGCAATT-TGCAAGATGGTC 14192.6 kb 136 15901-L17994 MAP3K12, ex 2 12q13.13 GCATCCAGAGTT-CGAGCTGACGAG 2479.8 kb 400 14405-L16087 CDK2, ex 1 12q13.2 CATTGTTTCAAG-TTGGCCAAATTG 1498.0 kb 154 15902-L17995 GLI1, ex 4 NM_005269.2; 421-422 ACTCGCGATGCA-CATCTCCAGGAG 4.8 kb 346 15908-L18001 GLI1, ex 11 NM_005269.2; 1598-1599 GGACCAGCTACA-TCAACTCCGGCC 47.1 kb 330 15907-L20363 DDIT3, ex 3 12q13.3 CCTCTCACTAGT-GCCAATGATGTG 229.1 kb 202 15903-L18385 TSPAN31, ex 2 12q14.1 TCCACATCATCG-GCGGAGTCATTG 2.8 kb 267 15904-L18392 CDK4, ex 8 NM_000075.2; 1062-1063 TGCTGACTTTTA-ACCCACACAAGC 2.7 kb 142 03173-L02512 CDK4, ex 3 NM_000075.2; 505-506 AACCCTGGTGTT-TGAGCATGTAGA 73.4 kb 324 16903-L20362 MIR26A2, ex 1 12q14.1 AGGCCTCACAGA-TGGAAACAGCCT 8000.2 kb 283 16186-L16821 HMGA2, ex 1 NM_003483.4; 357-358 CCGCCTAACATT-TCAAGGGACACA 3.2 kb 418 15074-L16832 HMGA2, ex 2 NM_003483.4; 962-963 GACCCAGGGGAA-GACCCAAAGGCA 10.5 kb 148 15075-L16838 HMGA2, ex 3 NM_003483.4; 1029-1030 AGCCACTGGAGA-AAAACGGCCAAG 76.8 kb 445 15086-L16849 HMGA2, ex 4 NM_003483.4; 1322-1323 CCAAGATGTAGT-TTCACTGCTACC 48.0 kb 427 15084-L16847 HMGA2, ex 5 NM_003483.4; 1217-1218 AGTGACCACTTA-TTCTGTATTGCC 2844.9 kb 338 02894-L20364 MDM2, ex 1 NM_002392.5; 133-134 CGAGATCCTGCT-GCTTTCGCAGCC 16.1 kb 370 00337-L18786 MDM2, ex 6 NM_002392.5: 691-692 GTACATCTGTGA-GTGAGAACAGGT 0.2 kb 190 07182-L06791 MDM2, ex 7 NM_002392.5; 768-769 GAGAAACCTTCA-TCTTCACATTTG 4.2 kb 262 07183-L18784 MDM2, ex 8 NM_002392.5; 877-878 GAAAACGCCACA-AATCTGATAGTA 531.2 kb 274 15905-L18393 YEATS4, ex 1 12q15 TATGTTCAAGAG-AATGGCCGAATT 16158.3 kb 208 14414-L16627 ALX1, ex 1 12q21.31 GTCTGCAGGCAA-ATGCGTGCAGGC 17195.4 kb 310 02340-L01834 IGF1, ex 2 12q23.2 AGGTAGAAGAGA-TGCGAGGAGGAC 9947.3 kb 409 12523-L13573 PTPN11, ex 1 12q24.13 CAGGAGGAAGCA-AGGATGCTTTGG 1947.6 kb 160 ± 05694-L05136 TBX5, ex 8 12q24.21 GTGAGGCAAAAA-GTGGCCTCCAAC 6633.1 kb 253 07717-L18390 HNF1A, ex 9 12q24.31 GCCTCAGTGTCT-GAGGTGAAGACC 9500.4 kb 226 09841-L18685 PIWIL1, ex 21 12q24.33 CAGAGAGCCAAA-TCTGTCACTGTC 501.3 kb 300 15906-L18397 RAN, ex 3 12q24.33 GTGTTTTTCAAC-AGCTTGTATTGG 1.7 kb 355 15909-L18002 RAN, ex 5 12q24.33 GTACTAATTCCC-ACAAATGTTTCT 2049.3 kb 238 02738-L18389 CHFR, ex 1 12q24.33 GGCGGCGGCGCT-CACCAAGAGCGG Reference probes on chr. 14, 15, 16, 18 and 19. 292 07017-L06628 RPGRIP1 14q11.2 CTACATCAGGAG-ACTTGCCAGTTA 166 14281-L15951 OCA2 15q13.1 GCCGCGATGAGA-CAGAGCATGATG 377 06216-L20365 TGFB1I1 16p11.2 CAGGAACTTAAT-GCCACTCAGTTC 385 05914-L05359 RNMT 18p11.21 TACAATGAACTT-CAGGAAGTTGGT 5669.9 kb 122 02844-L02274 NPC1 18q11.2 GACGAGTCTGTG-GATGAGGTCACA 216 14640-L18386 SDHAF1 19q13.12 AGCGTCTCTGCT-TAGCCGCGGTCA ± SNP rs147405081 and rs375955080 could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. The sequence targeted by this reference probe has been reported to be frequently affected by copy number changes in sarcoma samples. Data analysis should be performed with caution for this probe. SALSA probemix P323 CDK4-HMGA2-MDM2 Page 4 of 6
Note: Exon numbering may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA MLPA probemix P323-B1 CDK4-HMGA2-MDM2 sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P323-B1 CDK4-HMGA2-MDM2 (lot B1-0714). SALSA probemix P323 CDK4-HMGA2-MDM2 Page 5 of 6
Implemented Changes compared to the previous product description version(s). Version 07 (T08) 11 December 2015 - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Reference added on page 2. - Warning added in Table 1 and 2, 160 nt probe 05694-L05136. - Warning added in Table 1 and 2 about reference probe at 436 nt (05504-L04927). - Various minor layout changes. - Mapview locations removed from Table 2 and added to Table 1. - Ligation sites and transcript numbers indicated for several probes in Table 2. 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 6 of 6