MRC-Holland MLPA. Description version 13;

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1 SALSA MLPA probemix P027-C1 Uveal Melanoma Lot C1-0211: A large number of probes have been replaced by other probes in the same chromosomal regions as compared to previous lots, and several reference probes have been replaced/included.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): Damato B.E. et al. (2010) Genotypic profiling of 452 choroidal melanomas with Multiplex Ligation- Dependent Probe Amplification. Clin Cancer Res. 16(24): Dopierala J. et al. (2011) Genetic heterogeneity in uveal melanoma assessed by multiplex ligationdependent probe amplification. Invest Ophthalmol Vis Sci. 51(10): 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): Damato B. et al. (2009) Multiplex ligation-dependent probe amplification of uveal melanoma: correlation with metastatic death. Invest Ophthalmol Vis Sci. 50(7): More information Website : 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

2 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 nt, three DNA denaturation control fragments (Dfragments) 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 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: When in doubt, users should always verify the latest update of this product description ( 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

3 Table 1. SALSA MLPA P027-C1 Uveal Melanoma probemix Length Chromosomal position SALSA MLPA probe (nt) reference Chr 1 Chr 3 Chr 6 Chr 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 NOTCH2 probe L p * MLH1 probe L p * Reference probe L q MYC probe L q RUNX2 probe L p * MYC probe L q * Reference probe L q FHIT probe L p * RP1 probe L q * MIR128-2 probe L p MFN2 probe L p ± MME probe L q * CHL1 probe L p * Reference probe L p * GJB3 probe L p * BAP1 probe L p IGF2R probe L q RPE65 probe L p * CASR probe L q * Reference probe L q * PPARG probe L p CTGF probe L q NBL1 probe L p * C3ORF10 probe L p * Reference probe L p * ASAP1 probe L q * LZTS1 probe L p NRG1 probe L p * PROS1 probe L q FHIT probe L p OPA1 probe L q PTAFR probe L p * RBM5 probe L p * Reference probe L q ECI2 probe L p * VHL probe L p * Reference probe L q ROBO1 probe L p MUTYH probe L p CDKN1A probe L p * Reference probe L q * VHL probe L p * CTNNB1 probe L p DCDC2 probe L p * XPC probe L p * BAP1 probe L p * Reference probe L p * Reference probe L p16. * New in version C1 (from lot C onwards). SALSA probemix P027 Uveal Melanoma Page 3 of 7

4 Small change in length in version C1 (from lot C 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: 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: ) L04272 MFN2, ex 13 CTGGTGGACGAT-TACCAGATGGAC kb L03503 NBL1, ex 8 AGCTGCACAATT-TAATATATTCAA kb L01425 PTAFR, ex 4 CATCTTCATCGT-GTTCAGCTTCTT kb L17892 GJB3, ex 2 CCATGGGAGTGT-GTCAGGTGGAAG kb L03351 MUTYH, ex 5 CTCATACCATCT-ATTCAGAGACGT kb L19174 RPE65, ex 5 ATGCCCTTGTTA-ATGTCTACCCAG kb L02334 NOTCH2, ex 30 AAGCTGCAGACA-TCCGTAGGACAC Reference probes on 2p and 2q L15194 PEX13, 2p16.1 TGAGGATGACCA-TGTAGTTGCCAG kb L19277 EDAR, 2q12.3 AGAATCAAGGCT-TTTGTGATATGT 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: ) as well as the tumour suppressor genes VHL, at 3p25.3, and FHIT at 3p L17987 CHL1, ex 2 CCTAGGTGCTGT-AAACTGCAAACC kb L18089 C3ORF10, ex 3 AAAAGGTGAGAC-ACTCACCTAGAA 20.3 kb L14735 VHL, ex 2 CGTCAACATTGA-GAGATGGCACAA 3.3 kb L18091 VHL, ex 3 CCAAATGTGCAG-AAAGACCTGGAG kb L06480 PPARG, ex 3 ATACAACAAGGC-CATTTTCTCAAA kb L05573 XPC, ex 11 AGCAAGAGTGGT-GAGGCTTGGAGA kb L17989 MIR128-2 GAGAGTGAGTAG-CAGGTCTCACAG kb L20037 MLH1, ex 1 TCTAACGCGCAA-GCGCATATCCTT kb L18832 CTNNB1, ex 15b GCTGACTATCCA-GTTGATGGGCTG kb L18094 RBM5, ex 3 ATATGATGACTA-CCGAGACTATGA kb L19176 BAP1, ex 9 AACCTGATGGCA-GTGGTGCCCGAC 2.2 kb L20039 BAP1, ex 4 ATACGTCCGTGA-TTGATGATGATA kb L02212 FHIT, ex 5 GAGGACATGTCG-TTCAGATTTGGC kb L01781 FHIT, ex 4 CCTGCCTGCTTA-GACCCTCTATAA kb L03256 ROBO1, ex 29 ATATGGATACGG-ATGCGCCAGAAG kb L04685 PROS1, ex 7 TGTGAATGCCCC-GAAGGCTACAGA kb L19175 CASR, ex 6 CTCCATCGTGTT-TAAGGAAGTCGG kb L00069 MME, ex 21 CGTTGACTGGTG-GACTCAACAGTC kb L02708 OPA1, ex 29 TGAAGATGGTGA-GAAGAAGATTAA Reference probes on 4q and 5q SALSA probemix P027 Uveal Melanoma Page 4 of 7

5 L03831 GNRHR, 4q13.2 GGACTGGTCTAA-GCTGCTCAAGAT L00463 IL4, 5q31.1 ATCGACACCTAT-TAATGGGTCTCA 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) L11363 ECI2, ex 11 AAGGACTTGTTA-CTGAAGTTTTCC kb L18092 DCDC2, ex 9 GCAGAGAGGTCT-GAAACACGGGGG kb L18090 CDKN1A, ex 6 CGGCTGATCTTC-TCCAAGAGGAAG kb L02082 RUNX2, ex 3 GTTGTGATGCGT-ATTCCTGTAGAT kb L19276 CTGF, ex 5 ACCGAGCTAAAT-TCTGTGGAGTAT kb L20038 IGF2R, ex 3 TTCAACACAACA-GTGAGCTGTGAC p 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: ), ASAP1, at 8q24.21 (Ehlers J.P. et al. 2005, Clin Cancer Res, 11: ) and MYC, at 8q L19178 LZTS1, ex 3 GCTGCAGCGCAA-GAAGAACGAGGC kb L03575 NRG1, ex 10 CTGGGACAAGCC-ATCTTGTAAAAT kb L19172 RP1, ex 3 ATCCTGAGCTCT-GGAGCTGTGGTG kb L16789 MYC, ex 1 CTGGAACTTACA-ACACCCGAGCAA 4.3 kb L00145 MYC, ex 3 GAACGAGCTAAA-ACGGAGCTTTTT kb L18499 ASAP1, ex 29 TTCCTTTCAGGC-TGTCCTTCGATG Reference probes on 10p, 11p, 12q, 13q, 14q, 15q, 18p and 18q L07319 GATA3, 10p14 GGGGCAACCTCG-ACCCCACTGTGG L09031 SLC6A5, 11p15.1 TGTTTGCCTCCT-TTGTGTCTGTAC L16128 ALX1, 12q21.31 ATGACACCTTAT-TCTCACTCGCCT L19694 ATP7B, 13q14.3 GCAGGAGAGACA-GATCACAGCCAG L18088 BCL2L2, 14q11.2 GTGGCAGACTTT-GTAGGTTATAAG L10194 SPG11, 15q21.1 CCAGTGTAAGCA-GTATGCTATTGG L17745 RNMT, 18p11.21 TACAATGAACTT-CAGGAAGTTGGT kb L02274 NPC1, 18q11.2 GACGAGTCTGTG-GATGAGGTCACA 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

6 SALSA MLPA probemix P027-C1 Uveal Melanoma sample picture D y e S i g n a l 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 D y e S i g n a l 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

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