P323-B1 CDK4-HMGA2-MDM2

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1 SALSA MLPA probemix P323-B1 CDK4-HMGA2-MDM2 Lot B1-0714, B 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 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 Variations in the high-mobility group-a2 gene (HMGA2) are associated with idiopathic short stature. Pediatr Res. PMID: [Epub ahead of print] SALSA probemix P323 CDK4-HMGA2-MDM2 Page 1 of 6

2 More information Website : 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 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 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 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: 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

3 Table 1. SALSA MLPA P323-B1 CDK4-HMGA2-MDM2 probemix Length Chromosomal position MV location SALSA MLPA probe (nt) reference 12p 12q (HG18) 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 122 Reference probe L q Reference probe L q MAP3K12 probe L q «CDK4 probe L q HMGA2 probe L q GLI1 probe L q ± TBX5 probe L q Reference probe L q CCND2 probe L p Reference probe L q KRAS probe L p MDM2 probe L q Reference probe L q TSPAN31 probe L q ALX1 probe L q Reference probe L q PIWIL1 probe L q FOXM1 probe L p CHFR probe L q Reference probe L p HNF1A probe L q MDM2 probe L q CDK4 probe L q YEATS4 probe L q HMGA2 probe L q Reference probe L q RAN probe L q IGF1 probe L q CDKN1B probe L p MIR26A2 probe L q DDIT3 probe L q MDM2 probe L q GLI1 probe L q RAN probe L q KIF21A probe L q MDM2 probe L q Reference probe L p Reference probe L p CCND2 probe L p CDK2 probe L q PTPN11 probe L q «HMGA2 probe L q HMGA2 probe L q Reference probe L q HMGA2 probe L q Reference probe L q «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 rs and rs 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

4 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 L20366 PAX8 2q13 TTGCAGATGCTA-GGACACAAGAGA L06889 POU1F1 3p11.2 TCCTATACACCA-GCCTCTTCTGGC kb L04688 PROS1 3q11.1 CATTTAAATCCC-CAGCATAAATCA L04183 GNRHR 4q13.2 TGGAACATTACA-GTCCAATGGTAT L13645 IL4 5q31.1 ATCGACACCTAT-TAATGGGTCTCA L04927 RET 10q11.21 CCTGCAACTGCT-TCCCTGAGGAGG 12p chromosomal arm L18686 FOXM1, ex 4 12p13.33 CCATGATACAAT-TCGCCATCAACA kb L02516 CCND2, ex 1 NM_ ; AGACCAGTTTTA-AGGGGAGGACCG 29.9 kb L18979 CCND2, ex 5 NM_ ; TAACAGCCAAGA-AGCCTGCAGGAG kb L18978 CDKN1B, ex 1 12p13.1 CGCGCTCCTAGA-GCTCGGGCCGTG kb L11071 KRAS, ex 2 12p12.1 ATTTTGTGGACG-AATATGATCCAA kb 12q chromosomal arm L18394 KIF21A, ex 38 12q12 AGGCTCGCAATT-TGCAAGATGGTC kb L17994 MAP3K12, ex 2 12q13.13 GCATCCAGAGTT-CGAGCTGACGAG kb L16087 CDK2, ex 1 12q13.2 CATTGTTTCAAG-TTGGCCAAATTG kb L17995 GLI1, ex 4 NM_ ; ACTCGCGATGCA-CATCTCCAGGAG 4.8 kb L18001 GLI1, ex 11 NM_ ; GGACCAGCTACA-TCAACTCCGGCC 47.1 kb L20363 DDIT3, ex 3 12q13.3 CCTCTCACTAGT-GCCAATGATGTG kb L18385 TSPAN31, ex 2 12q14.1 TCCACATCATCG-GCGGAGTCATTG 2.8 kb L18392 CDK4, ex 8 NM_ ; TGCTGACTTTTA-ACCCACACAAGC 2.7 kb L02512 CDK4, ex 3 NM_ ; AACCCTGGTGTT-TGAGCATGTAGA 73.4 kb L20362 MIR26A2, ex 1 12q14.1 AGGCCTCACAGA-TGGAAACAGCCT kb L16821 HMGA2, ex 1 NM_ ; CCGCCTAACATT-TCAAGGGACACA 3.2 kb L16832 HMGA2, ex 2 NM_ ; GACCCAGGGGAA-GACCCAAAGGCA 10.5 kb L16838 HMGA2, ex 3 NM_ ; AGCCACTGGAGA-AAAACGGCCAAG 76.8 kb L16849 HMGA2, ex 4 NM_ ; CCAAGATGTAGT-TTCACTGCTACC 48.0 kb L16847 HMGA2, ex 5 NM_ ; AGTGACCACTTA-TTCTGTATTGCC kb L20364 MDM2, ex 1 NM_ ; CGAGATCCTGCT-GCTTTCGCAGCC 16.1 kb L18786 MDM2, ex 6 NM_ : GTACATCTGTGA-GTGAGAACAGGT 0.2 kb L06791 MDM2, ex 7 NM_ ; GAGAAACCTTCA-TCTTCACATTTG 4.2 kb L18784 MDM2, ex 8 NM_ ; GAAAACGCCACA-AATCTGATAGTA kb L18393 YEATS4, ex 1 12q15 TATGTTCAAGAG-AATGGCCGAATT kb L16627 ALX1, ex 1 12q21.31 GTCTGCAGGCAA-ATGCGTGCAGGC kb L01834 IGF1, ex 2 12q23.2 AGGTAGAAGAGA-TGCGAGGAGGAC kb L13573 PTPN11, ex 1 12q24.13 CAGGAGGAAGCA-AGGATGCTTTGG kb 160 ± L05136 TBX5, ex 8 12q24.21 GTGAGGCAAAAA-GTGGCCTCCAAC kb L18390 HNF1A, ex 9 12q24.31 GCCTCAGTGTCT-GAGGTGAAGACC kb L18685 PIWIL1, ex 21 12q24.33 CAGAGAGCCAAA-TCTGTCACTGTC kb L18397 RAN, ex 3 12q24.33 GTGTTTTTCAAC-AGCTTGTATTGG 1.7 kb L18002 RAN, ex 5 12q24.33 GTACTAATTCCC-ACAAATGTTTCT kb L18389 CHFR, ex 1 12q24.33 GGCGGCGGCGCT-CACCAAGAGCGG Reference probes on chr. 14, 15, 16, 18 and L06628 RPGRIP1 14q11.2 CTACATCAGGAG-ACTTGCCAGTTA L15951 OCA2 15q13.1 GCCGCGATGAGA-CAGAGCATGATG L20365 TGFB1I1 16p11.2 CAGGAACTTAAT-GCCACTCAGTTC L05359 RNMT 18p11.21 TACAATGAACTT-CAGGAAGTTGGT kb L02274 NPC1 18q11.2 GACGAGTCTGTG-GATGAGGTCACA L18386 SDHAF1 19q13.12 AGCGTCTCTGCT-TAGCCGCGGTCA ± SNP rs and rs 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

5 Note: Exon numbering may differ from literature! Complete probe sequences are available on request: Please notify us of any mistakes: 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

6 Implemented Changes compared to the previous product description version(s). Version 07 (T08) 11 December 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 L 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 Electropherogram picture(s) using the old MLPA buffer (replaced in December 2012) removed. Version 05 (48) - Warning added in Table 1, 142 nt probe L02512 and 418 nt probe L 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