SALSA MLPA probemix P027-C1 Uveal Melanoma Lot C1-0211: A large number of probes have been replaced by other probes in the same chromosomal regions as compared to previous lots, and several reference probes have been replaced/included. Details are in Table 1. As a result, the version number has been changed to C1. In addition, the 88 and 96nt control fragments have been replaced (QDX2). Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with mortality over 50% due to metastatic spread to the liver. Recent research suggests that uveal melanomas with monosomy 3 (50-60% of all uveal melanomas) represent a distinct pathological entity as compared to uveal melanomas with normal disomy 3, as monosomy of chromosome 3 strongly correlates with metastatic death in UM. Chromosome 6 aberrations probably constitute a second entry point in the process of carcinogenesis, while gains in 8q seem to appear later in the natural history of uveal melanomas - due to their higher frequency in larger tumors. Early detection of high risk UM patients would enable better screening for metastasis and for optimized therapy selection, and secondly to reassure patients with no loss of chromosome 3, which is associated with very good prognosis. This P027-C1 Uveal Melanoma MLPA probemix contains several probes on chromosomes 1p, 3, 6p and 8q (MYC region). In addition, it contains 12 reference probes detecting sequences in autosomal chromosome regions that are relatively quiet in uveal melanoma. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the above mentioned chromosomal regions in a DNA sample. Heterozygous deletions of probe 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, 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. 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). References for SALSA probemix P027 Uveal Melanoma Lake S.L. et al. (2011) Multiplex ligation-dependent probe amplification analysis of uveal melanoma with extraocular extension demonstrates heterogeneity of gross chromosomal abnormalities. Invest Ophthalmol Vis Sci. 52(8):5559-64. Damato B.E. et al. (2010) Genotypic profiling of 452 choroidal melanomas with Multiplex Ligation- Dependent Probe Amplification. Clin Cancer Res. 16(24):6083-92. Dopierala J. et al. (2011) Genetic heterogeneity in uveal melanoma assessed by multiplex ligationdependent probe amplification. Invest Ophthalmol Vis Sci. 51(10):4898-905. Lake S.L. et al. (2010) Whole-genome microarray detects deletions and loss of heterozygosity of chromosome 3 occurring exclusively in metastasizing uveal melanoma. Invest Ophthalmol Vis Sci. 51(10):4884-91. Damato B. et al. (2009) Multiplex ligation-dependent probe amplification of uveal melanoma: correlation with metastatic death. Invest Ophthalmol Vis Sci. 50(7):3048-55. 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 P027 Uveal Melanoma Page 1 of 7
Data analysis The P027-C1 probe mix contains 50 MLPA probes with amplification products between 122 and 499 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 (Dfragments) 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 uveal melanoma, 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://projects.tcag.ca/variation. When in doubt, users should always verify the latest update of this product description (www.mlpa.com) and the scientific literature when interpreting their findings. This probemix was developed by 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 designer could be made a coauthor. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P027 Uveal Melanoma Page 2 of 7
Table 1. SALSA MLPA P027-C1 Uveal Melanoma probemix Length Chromosomal position SALSA MLPA probe (nt) reference Chr 1 Chr 3 Chr 6 Chr 8 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 130 Reference probe 00797-L00463 5q31 136 NOTCH2 probe 02867-L02334 1p12 140 * MLH1 probe 15288-L20037 3p22.2 148 * Reference probe 04445-L03831 4q13 154 MYC probe 00580-L00145 8q24.21 160 RUNX2 probe 02611-L02082 6p21.1 166 * MYC probe 15894-L16789 8q24.21 172 * Reference probe 03237-L19694 13q14 178 FHIT probe 02292-L02212 3p14.2 184 * RP1 probe 16641-L19172 8q12.1 190 * MIR128-2 probe 15896-L17989 3p22.3 196 MFN2 probe 04888-L04272 1p36.22 202± MME probe 00487-L00069 3q25.2 207 * CHL1 probe 14147-L17987 3p26.3 211 * Reference probe 07634-L07319 10p14 220 * GJB3 probe 13671-L17892 1p34.3 226 * BAP1 probe 16643-L20039 3p21.1 233 IGF2R probe 02798-L20038 6q25.3 241 RPE65 probe 03601-L19174 1p31.2 247 * CASR probe 05708-L19175 3q21.1 255 * Reference probe 03187-L18088 14q11 261 * PPARG probe 06900-L06480 3p25.2 266 CTGF probe 04745-L19276 6q23.2 274 NBL1 probe 04148-L03503 1p36.13 283 * C3ORF10 probe 15895-L18089 3p25.3 292 * Reference probe 08936-L09031 11p15 299 * ASAP1 probe 16241-L18499 8q24.21 310 * LZTS1 probe 02552-L19178 8p21.3 319 NRG1 probe 04239-L03575 8p12 328 * PROS1 probe 05297-L04685 3q11.1 337 FHIT probe 02290-L01781 3p14.2 346 OPA1 probe 03271-L02708 3q29 355 PTAFR probe 02267-L01425 1p35.3 364 * RBM5 probe 15897-L18094 3p21.31 373 * Reference probe 09779-L10194 15q21 382 ECI2 probe 10252-L11363 6p25.2 391 * VHL probe 13322-L14735 3p25.3 400 * Reference probe 14423-L16128 12q21 409 ROBO1 probe 04603-L03256 3p12.3 418 MUTYH probe 03964-L03351 1p34.1 427 CDKN1A probe 00585-L18090 6p21.2 437 * Reference probe 12790-L19277 2q12 445 * VHL probe 15899-L18091 3p25.3 454 * CTNNB1 probe 16407-L18832 3p22.1 463 DCDC2 probe 10253-L18092 6p22.3 474 * XPC probe 06118-L05573 3p25.1 481 * BAP1 probe 16644-L19176 3p21.1 490 * Reference probe 14909-L17745 18p11 499 * Reference probe 09870-L15194 2p16. * New in version C1 (from lot C1-0211 onwards). SALSA probemix P027 Uveal Melanoma Page 3 of 7
Small change in length in version C1 (from lot C1-0211 onwards). No change in the sequence detected. ± This probe has been reported to be more variable. 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. Table 2. P027-C1 probes arranged according to chromosomal location Length (nt) Loss of 1p arm SALSA MLPA probe Gene, exon Partial sequence (24 nt adjacent to ligation site) Distance to next probe MV location (HG18) Studies have shown a correlation between loss of 1p arm and poor prognosis in UM, in particular for patients with loss of both 1p arm and loss of chromosome 3 (Kilic E. et al. 2005, IOVS, 46:2253-2257). 196 04888-L04272 MFN2, ex 13 CTGGTGGACGAT-TACCAGATGGAC 7869.2 kb 01-011.987183 274 04148-L03503 NBL1, ex 8 AGCTGCACAATT-TAATATATTCAA 8493.1 kb 01-019.856370 355 02267-L01425 PTAFR, ex 4 CATCTTCATCGT-GTTCAGCTTCTT 6671.0 kb 01-028.349503 220 13671-L17892 GJB3, ex 2 CCATGGGAGTGT-GTCAGGTGGAAG 10551.2 kb 01-035.020515 418 03964-L03351 MUTYH, ex 5 CTCATACCATCT-ATTCAGAGACGT 23111.1 kb 01-045.571725 241 03601-L19174 RPE65, ex 5 ATGCCCTTGTTA-ATGTCTACCCAG 51581.6 kb 01-068.682851 136 02867-L02334 NOTCH2, ex 30 AAGCTGCAGACA-TCCGTAGGACAC 01-120.264421 Reference probes on 2p and 2q 499 09870-L15194 PEX13, 2p16.1 TGAGGATGACCA-TGTAGTTGCCAG 47753.3 kb 02-061.126370 437 12790-L19277 EDAR, 2q12.3 AGAATCAAGGCT-TTTGTGATATGT 02-108.879653 Loss of chromosome 3 Monosomy of chromosome 3 is the most common chromosomal abnormality detected in ~50% of UM patients and it has strong association with short survival (Prescher G. et al. 1996, Lancet, 347:1222 5). Several different candidate genes have been reported, including BAP1 (Harbour JW. Et al. 2010, Science, 330:1410-3), RBM5, PPARG, ROBO1 (Lake S. et al. 2010, IOVS, 51:4884-91) as well as the tumour suppressor genes VHL, at 3p25.3, and FHIT at 3p.14.2. 207 14147-L17987 CHL1, ex 2 CCTAGGTGCTGT-AAACTGCAAACC 9881.7 kb 03-000.261254 283 15895-L18089 C3ORF10, ex 3 AAAAGGTGAGAC-ACTCACCTAGAA 20.3 kb 03-010.142937 391 13322-L14735 VHL, ex 2 CGTCAACATTGA-GAGATGGCACAA 3.3 kb 03-010.163233 445 15899-L18091 VHL, ex 3 CCAAATGTGCAG-AAAGACCTGGAG 2162.3 kb 03-010.166565 261 06900-L06480 PPARG, ex 3 ATACAACAAGGC-CATTTTCTCAAA 1839.9 kb 03-012.328889 474 06118-L05573 XPC, ex 11 AGCAAGAGTGGT-GAGGCTTGGAGA 21592.2 kb 03-014.168823 190 15896-L17989 MIR128-2 GAGAGTGAGTAG-CAGGTCTCACAG 1248.8 kb 03-035.760989 140 15288-L20037 MLH1, ex 1 TCTAACGCGCAA-GCGCATATCCTT 4246.0 kb 03-037.009760 454 16407-L18832 CTNNB1, ex 15b GCTGACTATCCA-GTTGATGGGCTG 8848.9 kb 03-041.255710 364 15897-L18094 RBM5, ex 3 ATATGATGACTA-CCGAGACTATGA 2310.8 kb 03-050.104592 481 16644-L19176 BAP1, ex 9 AACCTGATGGCA-GTGGTGCCCGAC 2.2 kb 03-052.415358 226 16643-L20039 BAP1, ex 4 ATACGTCCGTGA-TTGATGATGATA 8080.2 kb 03-052.417524 178 02292-L02212 FHIT, ex 5 GAGGACATGTCG-TTCAGATTTGGC 285.0 kb 03-060.497686 337 02290-L01781 FHIT, ex 4 CCTGCCTGCTTA-GACCCTCTATAA 17967.1 kb 03-060.782679 409 04603-L03256 ROBO1, ex 29 ATATGGATACGG-ATGCGCCAGAAG 16352.6 kb 03-078.749746 328 05297-L04685 PROS1, ex 7 TGTGAATGCCCC-GAAGGCTACAGA 28375.1 kb 03-095.102326 247 05708-L19175 CASR, ex 6 CTCCATCGTGTT-TAAGGAAGTCGG 32891.5 kb 03-123.477457 202 00487-L00069 MME, ex 21 CGTTGACTGGTG-GACTCAACAGTC 38498.7 kb 03-156.368991 346 03271-L02708 OPA1, ex 29 TGAAGATGGTGA-GAAGAAGATTAA 03-194.867681 Reference probes on 4q and 5q SALSA probemix P027 Uveal Melanoma Page 4 of 7
148 04445-L03831 GNRHR, 4q13.2 GGACTGGTCTAA-GCTGCTCAAGAT 04-068.303935 130 00797-L00463 IL4, 5q31.1 ATCGACACCTAT-TAATGGGTCTCA 05-132.037610 Gain of 6p and loss of 6q Gain of 6p, often due to isochromosome 6p, is associated with good prognosis in UM (White V.A. et al. 1998, Cancer, 83:354-9). 382 10252-L11363 ECI2, ex 11 AAGGACTTGTTA-CTGAAGTTTTCC 20250.7 kb 06-004.062570 463 10253-L18092 DCDC2, ex 9 GCAGAGAGGTCT-GAAACACGGGGG 12448.3 kb 06-024.313233 427 00585-L18090 CDKN1A, ex 6 CGGCTGATCTTC-TCCAAGAGGAAG 8736.7 kb 06-036.761516 160 02611-L02082 RUNX2, ex 3 GTTGTGATGCGT-ATTCCTGTAGAT 86814.0 kb 06-045.498265 266 04745-L19276 CTGF, ex 5 ACCGAGCTAAAT-TCTGTGGAGTAT 28037.8 kb 06-132.312257 233 02798-L20038 IGF2R, ex 3 TTCAACACAACA-GTGAGCTGTGAC 06-160.350052 8p loss and 8q gain 8p loss and 8q gain, often due to isochromosome 8q, are associated with poor prognosis in UM. Several potential target genes have been suggested e.g. LZTS1 gene, at 8p21.2 (Onken M. et al. 2008, Clin Cancer Res, 14:3737-45), ASAP1, at 8q24.21 (Ehlers J.P. et al. 2005, Clin Cancer Res, 11:3609-13) and MYC, at 8q24.21. 310 02552-L19178 LZTS1, ex 3 GCTGCAGCGCAA-GAAGAACGAGGC 12553.1 kb 08-020.151894 319 04239-L03575 NRG1, ex 10 CTGGGACAAGCC-ATCTTGTAAAAT 22992.2 kb 08-032.705011 184 16641-L19172 RP1, ex 3 ATCCTGAGCTCT-GGAGCTGTGGTG 73120.6 kb 08-055.697238 166 15894-L16789 MYC, ex 1 CTGGAACTTACA-ACACCCGAGCAA 4.3 kb 08-128.817870 154 00580-L00145 MYC, ex 3 GAACGAGCTAAA-ACGGAGCTTTTT 2314.0 kb 08-128.822148 299 16241-L18499 ASAP1, ex 29 TTCCTTTCAGGC-TGTCCTTCGATG 08-131.136182 Reference probes on 10p, 11p, 12q, 13q, 14q, 15q, 18p and 18q 211 07634-L07319 GATA3, 10p14 GGGGCAACCTCG-ACCCCACTGTGG 10-008.145969 292 08936-L09031 SLC6A5, 11p15.1 TGTTTGCCTCCT-TTGTGTCTGTAC 11-020.585664 400 14423-L16128 ALX1, 12q21.31 ATGACACCTTAT-TCTCACTCGCCT 12-084.219130 172 03237-L19694 ATP7B, 13q14.3 GCAGGAGAGACA-GATCACAGCCAG 13-051.483424 255 03187-L18088 BCL2L2, 14q11.2 GTGGCAGACTTT-GTAGGTTATAAG 14-022.846841 373 09779-L10194 SPG11, 15q21.1 CCAGTGTAAGCA-GTATGCTATTGG 15-042.690363 490 14909-L17745 RNMT, 18p11.21 TACAATGAACTT-CAGGAAGTTGGT 5669.9 kb 18-013.724478 122 02844-L02274 NPC1, 18q11.2 GACGAGTCTGTG-GATGAGGTCACA 18-019.394343 Note: 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 probemix P027 Uveal Melanoma Page 5 of 7
SALSA MLPA probemix P027-C1 Uveal Melanoma sample picture 2 59.6 4 60000 1 38.2 4 2 47.0 9 2 91.6 9 41 9.68 50000 8 6.43 96.4 6 1 69.9 4 1 88.6 0 1 51.3 5 2 05.5 0 24 0.26 2 66.3 8 25 4.55 2 32.4 0 2 9 9.2 3 4 83.1 5 40000 1 05.4 0 1 44.6 8 2 18.4 0 1 1 9.4 4 9 1.3 4 1 6 3.1 6 13 3.21 15 8.73 1 8 1.8 4 20 0.18 22 5.27 1 27.7 4 1 95.4 621 0.62 27 3.27 28 2.34 3 28.9 3 3 1 7.7 9 40 9.28 37 4.37 3 91.5 5 3 55.9 1 40 0.52 4 29.1 0 4 98.1 2 49 1.22 47 4.33 4 6 4.4 9 17 6.92 3 09.9 2 43 8.72 30000 3 45.1 5 3 64.2 0 1 0 0.6 7 3 37.2 5 4 4 5.3 2 3 83.4 9 4 54.4 1 20000 D y e S i g n a l 10000 0 100 150 200 250 300 350 400 450 500 Size (nt) Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P027-C1 Uveal Melanoma (lot C1-0211). The old MLPA buffer (replaced in December 2012) was used. Vials with the old MLPA buffer have a white label. 70000 1 3 8.22 60000 29 9.2 6 16 9.9 4 1 8 8.63 24 0.3 0 50000 1 19.3 9 1 4 4.64 16 3.1 4 2 0 5.56 2 32.4 4 26 6.4 1 2 5 4.60 3 1 7.78 40000 1 3 3.18 1 27.7 0 15 8.7 1 1 8 1.85 2 2 5.33 1 9 5.49 2 10.6 8 2 00.2 4 21 8.4 7 25 9.6 9 2 8 2.37 2 4 7.15 27 3.3 0 30 9.9 2 3 55.8 6 40 0.4 7 4 2 9.06 4 38.6 9 1 5 1.31 17 6.9 2 2 91.7 1 47 4.3 7 30000 20000 96.4 3 8 6.4 2 1 05.3 3 9 1.31 3 28.8 9 34 5.1 1 3 64.1 5 39 1.4 9 3 7 4.32 41 9.6 4 3 3 7.22 3 8 3.45 4 0 9.26 45 4.4 0 4 45.3 0 4 98.1 6 48 3.2 0 4 64.5 4 4 91.2 6 1 0 0.60 D y e S i g n a l 10000 0 100 150 200 250 300 350 400 450 500 Size (nt) Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P027-C1 Uveal Melanoma (lot C1-0211). The new MLPA buffer (introduced in December 2012) was used. Vials with the new MLPA buffer have a yellow label. SALSA probemix P027 Uveal Melanoma Page 6 of 7
Implemented Changes compared to the previous product description version(s). Version 13 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 12 (48) - Various textual changes. Version 11 (46) - New reference articles added for P027 on page 1. Version 10 (46) - Product description adapted to a new version (lot number added, changes in Table 1 and 2, new picture included). - New reference articles added for P027 probemix on page 1. - Warning added on page 2 about consequences of low tumour cell percentage for MLPA analysis. - Information on prognostic relevance of target chromosomal regions added in Table 2. - Warning added in Table 1, 202 nt probe (00487-L00069). Version 09 (44) - Various minor textual changes on page 1. - Minor changes in the data analysis section on page 2. - Tables have been numbered. - Complete probe sequences are available on request note have been added on page 5. SALSA probemix P027 Uveal Melanoma Page 7 of 7