SALSA MLPA probemix P383-A1 T-ALL Lot A

Transcription

1 SALSA MLPA probemix P383-A1 T-ALL Lot A T-lineage acute lymphoblastic leukaemia (T-ALL) is a clonal malignant disorder of immature T-cells, which accounts for about 15% of paediatric and 25% of adult ALL cases. Although prognosis has improved due to effective treatment protocols, still around 25% of paediatric and 50-70% of adult patients experience relapse. T-ALL is genetically heterogeneous, and comprises multiple distinct subtypes defined by chromosomal rearrangements involving usually one of the T-cell receptor loci, but also non-tcr-driven translocations are detected. In addition, genome-wide characterisation has identified in recent years several novel amplifications, deletions and point mutations in T-ALL samples that are of interest to better define T- ALL subgroups. This P383-A1 T-ALL probemix contains 56 probes for 13 different chromosomal regions, which are suggested to be of diagnostic and/or prognostic importance in T-ALL. These chromosomal areas and genes are: STIL- TAL1 (1p33), LEF1 (4q25), CASP8AP2 (6q15), MYB (6q23.3), EZH2 (7q36.1) CDKN2A/B+MTAP+MLLT3 (9p21.3), NUP214-ABL1 (9q34.1), PTEN (10q23.31), LMO1 (11p15.4), LMO2 (11p13), NF1+SUZ12 (17q11.2), PTPN2 (18p11.21) and PHF6 (Xq26.2). 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 recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Deletions of a probe s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding probe amplification product in males, whereas female heterozygotes are recognisable by a 35-50% reduction in relative peak height. Note that a mutation or polymorphism (e.g. SNP) 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, single deletions and duplications detected by MLPA should always be confirmed by other methods or by MLPA probemixes with higher resolution in the gene or chromosomal area of interest. 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). Related SALSA probemixes P202 IKZF1 (IKAROS): Contains probes for every exon of IKZF1 gene and also includes probes for IKZF2, IKZF3 and 9p21.3 region. P327 iamp21: Probes for RUNX1 and other chromosome 21 genes. P329 CLRF2: Contains probes for CRLF2, CSF2RA, IL3RA region in Xp22.33 PAR region, involved in ALL. P335 ALL-IKZF1: Contains probes for IKZF1, CDKN2A/2B, EBF1, Xp22 PAR-region, PAX5, ETV6, BTG1 and RB1. P377 Hematologic malignancies: Contains probes to screening DNA samples for the most significant copy number changes associated with ALL, AML, CLL, CML, MDS and lymphomas. References for SALSA probemix P383 T-ALL See detailed information and references on included chromosomal areas and genes in Table 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 SALSA probemix P383 T-ALL Page 1 of 9

2 Data analysis The P383-A1 T-ALL probemix contains 56 MLPA probes with amplification products between 130 nt and 504 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 robust method, as gains and losses are frequent in tumours. (1) Intra-sample normalisation should be performed by dividing the signal of each probe by the signal of every other probe* in that sample, thus creating as many ratios per probe as there are other 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. * We recommend to treat each probe as a reference probe with the exception of the CDKN2A/B, ABL1, NUP214 and Xq probes. 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. For analysis of the Xq26 probes we recommend to use reference samples that are all from the same gender. 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. 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 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 P383 T-ALL Page 2 of 9

3 Table 1. SALSA MLPA P383-A1 T-ALL probemix Probe location Length (nt) SALSA MLPA probe Chromosomal Exon MV location band (hg18/ncbi36) 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 130 STIL probe L p33 Exon PHF6 probe L23888 Xq26.2 Exon 1 X TAL1 probe L p33 Exon «MYB probe L q23.3 Exon RAG2 probe L p12 Exon «MLLT3 probe L p21.3 Exon PHF6 probe L23843 Xq26.2 Exon 3 X CASP8AP2 probe L q15 Exon «MLLT3 probe L p21.3 Exon Ж «PTPN2 probe SP0316-L p11.21 Exon CDKN2A probe L p21.3 Exon 2a SH2D1A probe L05559 Xq25 Exon 1 X LMO2 probe L p13 Exon ARHGEF6 probe L02296 Xq26.3 Exon 19 X Ж «PTPN2 probe SP0318-L p11.21 Exon PTEN probe L q23.31 Exon «LEF1 probe L q25 Exon PHF6 probe L23884 Xq26.2 Exon 7 X PTEN probe L q23.31 Exon Ж PTPN2 probe SP0317-L p11.21 Exon Ж LMO1 probe SP0381-L p15.4 Exon «NF1 probe L q11.2 Exon CDKN2B probe L p21.3 Exon EZH2 probe L q36.1 Exon 4b CASP8AP2 probe L q15 Exon STIL probe L p33 Exon CDKN2A probe L p21.3 Exon ABL1 probe L q34.12 Exon MYB probe L q23.3 Exon 16b AHI1 probe L q23.3 Exon EZH2 probe L q36.1 Exon «SUZ12 probe L q11.2 Exon ABL1 probe L q34.12 Exon «MYB probe L q23.3 Exon MTAP probe L p21.3 Exon Ж «LMO1 probe SP0380-L p15.4 Exon RAG2 probe L p12 Exon NUP214 probe L q34.13 Exon TAL1 probe L p33 Exon LEF1 probe L q25 Exon LMO2 probe L p13 Exon PHF6 probe L24481 Xq26.2 Exon 10c X «NF1 probe L q11.2 Exon CASP8AP2 probe L q15 Exon «LEF1 probe L q25 Exon CD44 probe L p13 Exon CASP8AP2 probe L q15 Exon PTEN probe L q23.31 Exon STIL probe L p33 Exon LEF1 probe L q25 Exon PTPN2 probe L p11.21 Exon 10b SALSA probemix P383 T-ALL Page 3 of 9

4 Probe location Length (nt) SALSA MLPA probe Chromosomal Exon MV location band (hg18/ncbi36) 469 Ж EZH2 probe SP0635-L q36.1 Exon PTEN probe L q23.31 Exon SLC1A2 probe L p13 Exon NUP214 probe L q34.13 Exon SUZ12 probe L q11.2 Exon Ж This probe consists of three parts and has two ligation sites. «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. Flanking probe. Included to facilitate determination of the extend of a deletion / duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition being tested. 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 probemix P383 T-ALL Page 4 of 9

5 Table 2. P383 probes arranged according to chromosomal location Length SALSA MLPA Partial sequence (24 nt Distance to Gene /Exon Ligation site (nt) probe adjacent to ligation site) next probe STIL-TAL1 fusion, at 1p33 Submicroscopic interstitial deletions (~90kb, starting at chr1: and ending at chr1: ) in the 5' UTR region of TAL1 gene and last STIL exons, lead to a STIL-TAL1 fusion gene and increased expression of TAL1 (Brown L et al EMBO J. 9: ). STIL-TAL1 fusion is found in 10-30% of T-ALL with a normal karyotype and fusion seems to be associated with favourable outcome in T-ALL (Cave H et al Blood. 103:442-50). ^ A decreased signal of the probes for STIL exons 6 and 12 is indicative for the formation of a STIL-TAL1 fusion gene L23860 TAL1, ex 4 NM_ ; GCAGCAGAATGT-GAACGGGGCCTT 9.4 kb L23841 TAL1, ex 1 NM_ ; CCCTGGACTGGT-TGGTCGGGGACA 51.5 kb 447 ^ L23865 STIL, ex 12 NM_ ; TCTTTTGAGACA-CTGCAAAGTAAG 19.1 kb 274 ^ L16883 STIL, ex 6 NM_ ; CTGCTTTCCCTA-AGAGTTCATATC 14.0 kb L18099 STIL, ex 1 NM_ ; CAACGTCCCAGA-GGCGGGGCCGGA - LEF1 gene, at 4q25 Mono/biallelic microdeletions of LEF1 are detected in 11% of children with T-ALL and inactivation seems to be correlating with better survival and younger age at diagnosis. Based on array CGH results the first exons of LEF1 are deleted most commonly (Gutierrez A et al Blood. 115: ) L23849 LEF1, ex 13 NM_ ; CTAGAGACGCTG-ATCCATAAAGAC 33.1 kb L23859 LEF1, ex 7 NM_ ; TATCCCATCACG-GGTGGATTCAGG 82.0 kb 417 «15968-L18120 LEF1, ex 4 NM_ ; TATGAATAACGA-CCCATACATGTC 4.6 kb 220 «12507-L24045 LEF1, ex 1 NM_ ; GCATCCGGGGTA-ACTACAGTGGAG - CASP8AP2 gene, at 6q15 Deletion of 6p , including the CASP8AP2 gene, was detected in 12% of T-ALL patients and is suggested to associate with poor early treatment response (Remke M et al Blood. 114: ) L23844 CASP8AP2, ex 1 NM_ ; GTAGTCCCCCGA-GTGGAGGTCGGC 16.6 kb L18109 CASP8AP2, ex 2 NM_ ; GGAAATAGGATC-ATGGCAGCAGAT 10.5 kb L23854 CASP8AP2, ex 6 NM_ ; TCTTAAGAAGAA-TATTTCAGCACT 16.8 kb L24482 CASP8AP2, ex 10 NM_ ; TTTCACATCAGC-ATGTTCAGCTTG kb to MYB gene MYB gene, at 6q23.3 Duplication of the MYB oncogene is detected in 8-15% of individuals with T-ALL (Clappier E et al Blood.110: ; O Neil J et al J Exp Med. 204: ). In most cases also the 3 end of the AHI1 gene is duplicated with MYB. The submicroscopic size of the aberration makes this duplication undetectable by conventional cytogenetics/fish in most cases (Lahortiga I et al Nat Genet. 39:593-5). MYB is a potential therapeutic target in T-ALL, although the prognostic/clinical association is still unclear due to small number of reported cases. 147 «12500-L18025 MYB, ex 2 NM_ ; TGAGGACTTTGA-GATGTGTGACCA 6.4 kb 326 «00441-L24480 MYB, ex 6 NM_ ; ATGCGTCGGAAG-GTCGAACAGGAA 26.1 kb L20654 MYB, ex 16b NM_ ; CATTTAATCCAG-ATTGTAAATGCT 66.9 kb L18112 AHI1, ex 30 NM_ ; GAAAACACTGTA-ATTGCTACTCAG - EZH2 gene, at 7q36.1 EZH2 gene has been shown to be inactivated by loss-of function mutations and deletions in T-ALL patients (Ntziachristos P et al Nat Med. 18: ; Zhang J et al Nature. 481: ). Moreover, loss of EZH2 in hematopoietic stem cells has been shown to cause aggressive T-ALL in mice (Simon C et al Genes Dev. 26: ). 469 Ж SP0635- EZH2, ex 21 NM_ ; and TTTTGCAATAAT-44nt spanning 7.6 kb L oligo-ttgtccttgttg L23021 EZH2, ex 15 NM_ ; TGCCCTTGTGTG-ATAGCACAAAAT 31.6 kb L23383 EZH2, ex 4b NM_ ; CAAGAATGGAAA-CAGCGAAGGATA - CDKN2A/B, MTAP and MLLT3 genes, at 9p21.3 CDKN2A/B inactivation is found in 50-60% of T-ALL cases. In a considerable number of patient samples, this deletion is submicroscopic and not detectable by conventional cytogenetics or by FISH (Usvasalo A et al 2008 Leukemia Res. 32: ; Sulong S et al Blood. 113:100-7). Another suggested target gene in the chromosomal region is MLLT3, which is deleted in 26% of T-ALL patients (Mullighan CG et al. Nature. 446:758-64). CDKN2A/B deletions are suggested to associate with poor outcome in both pediatric and adult T-ALL (Fizzotti M et al Blood. 85: ; Yamada Y et al J Clin Oncol. 15: ). 170 «16058-L23880 MLLT3, ex 7 NM_ ; GATAACGACAAT-GACTCTGAAATG kb 157 «12533-L23842 MLLT3, ex 1 NM_ ; TGTCCGCCATCT-ACCCTCCGCTCC kb L22421 MTAP, ex 1 NM_ ; GCCCACTGCAGA-TTCCTTTCCCGT kb L21990 CDKN2A, ex 5 NM_ ; 33nt before ex 5 TTGACCTCAGGT-TTCTAACGCCTG 7.0 kb L19013 CDKN2A, ex 2a NM_ ; 102nt before ex 2a CACGCCTTTGCT-GGCAGGCGGGGG 30.6 kb L23887 CDKN2B, ex 2 NM_ ; GCCTGTCTGAGA-CTCACAGGAAGG kb to ABL1 gene SALSA probemix P383 T-ALL Page 5 of 9

6 Length SALSA MLPA Partial sequence (24 nt Distance to Gene /Exon Ligation site (nt) probe adjacent to ligation site) next probe NUP214-ABL1 fusion gene, at 9q34.12-q13 Amplification of NUP214-ABL1 fusion gene has been observed in ~6% of T-ALL and it associates with aggressive course of disease (Craux C et al Nat Gen. 36:1084-9; Craux C et al Leukemia. 23:125-33). Detection of NUP214-ABL1 fusion amplification is potentially both of prognostic and clinical significance, since patients with NUP214-ABL1 fusion amplification could benefit from treatment with imatinib L24043 ABL1, ex 4 NM_ ; GCCCAAACCAAA-AATGGCCAAGGC 29.2 kb L24479 ABL1, ex 12 NM_ ; TCGAAAAGAGCG-AGGTCCCCCGGA kb L23891 NUP214, ex 2 NM_ ; AGAATCTTTGAC-TCCCCTGAGGAA 47.9 kb L24044 NUP214, ex 23 NM_ ; TCTGCTAGCAAA-ATTATTCCTCAA - PTEN gene, at 10q23.31 PTEN deletions are associated with early treatment failure and may contribute to increased resistance to chemotherapy. PTEN mutations in T-ALL seem to cluster in exon 7 and PTEN deletions occur in ~9% of T-ALL patients (Gutierrez A et al Blood. 114:647-50; Jotta P et al Leukemia. 24:239-42; Zuurbier L et al Haematologica. 97: ) L17760 PTEN, ex 1 NM_ ; CCTGCAGAAGAA-GCCCCGCCACCA 93.5 kb L23847 PTEN, ex 7 NM_ ; ACACGACGGGAA-GACAAGTTCATG 0.1 kb L23864 PTEN, ex 7 NM_ ; 3nt after ex 7 TAAAAAAGGTTT-GTACTTTACTTT 7.6 kb L17520 PTEN, ex 9 NM_ ; reverse AGAGAATTGTTC-CTATAACTGGTA - LMO1 gene, at 11p15.4 LMO1 is a transcriptional regulator, which is involved in translocations between TRB (7q34) and TRA/D (14q11) leading in overexpression of LMO1. More recently LMO1 has been recognized to be amplified and acting as a oncogene in neuroblastoma (Wang K et al Nature. 469:216-20). 343 Ж«16717-SP0380- L Ж SP0381- L23885 LMO1, ex 5 LMO1, ex 3 NM_ ; and reverse NM_ ; and reverse CCTCATAGTCCA-36nt spanning oligo-tgtctcccacac GTACTTGTCCAA-36nt spanning oligo-acagcccgcaca SALSA probemix P383 T-ALL Page 6 of kb kb to LMO2 gene LMO2 gene, at 11p13 Deletion of 11p13, including exon 1 of LMO2 gene, activates LMO2 expression in 4% of pediatric T-ALL patients by fusing RAG2 exon 1 to exon 2 of LMO2 (Van Vlierberghe P et al Blood. 108: ). A decreased signal of the probes for LMO2 ex 1, CD44 and SLC1A2 is indicative for the formation of a LMO2-RAG2 fusion L23846 LMO2, ex 5 NM_ ; ACATAGCATCCA-AGTGGCATAATT 33.0 kb L23858 LMO2, ex 1 NM_ ; AGCACATCTGGT-GTGAGAGAGCTC kb L23838 CD44, ex 19 NM_ ; CAGAAACTCCAG-ACCAGTTTATGA 36.4 kb L23892 SLC1A2, ex 13 NM_ ; AGTGCATGAAGA-TATTGAAATGAC kb L23889 RAG2, ex 3 NM_ ; GCATAACCATGT-CAAACTGAAGCC 4.1 kb L23853 RAG2, ex 1 NM_ : GTTTAGCGGCAA-AGATTCAGAGAG - NF1 and SUZ12 genes, at 17q11.2 NF1 is deleted in ~11% of T-ALL patients and NF1 deletion may correlate with poor response to induction therapy (Balgobind BV et al Blood. 111: ; Matteucci C et al Br J Heamatol. 149:70-8). Recently, also SUZ12 gene has been shown to be inactivated by loss-of function mutations and deletions in T-ALL patients (Ntziachristos P et al Nat Med. 18: ; Zhang J et al Nature. 481:157-63). 250 «03849-L18072 NF1, ex 26 NM_ ; TGAGGCACTGTA-CGGTCCTTGCAA kb 400 «02530-L23856 NF1, ex 58 NM_ ; GCCACTGTAACA-GTGGACGAACTC kb L23848 SUZ12, ex 10 NM_ ; CATCAGGAAAAC-AAGCCTGGTTCA 8.4 kb 311 «18705-L23805 SUZ12, ex 15 NM_ ; AGAAGTGATGAA-ACTCTGGAATCT PTPN2 gene, at 18p11.21 PTPN2 deletion is detected in ~6% T-ALL patients. Moreover, PTPN2 has been identified as a negative regulator of NUP214- ABL1 and JAK1 kinase activity (Kleppe M et al Nat Gen. 42:530-35; Kleppe M et al Blood. 117: ) L23850 PTPN2, ex 10b NM_ ; TCTGCACTAGTA-ACTGACAGTGCT 36.8 kb 208 Ж«18549-SP0318- L Ж SP0317- L Ж«15951-SP0316- L23881 PTPN2, ex 4 PTPN2, ex 2 PTPN2, ex 1 NM_ ; and NM_ ; and NM_ ; and TCACAGGGTCCA-27nt spanning oligo-cttatggtttgg AAGTTTCCAGAA-36nt spanning oligo-tgtaagtacttg CTCGCTCCCGCA-33nt spanning oligo-gagttggatact 28.2 kb 25.0 kb PHF6 gene, at Xq26.2 PHF6 inactivation is detected in 16% and 38% of T-ALL pediatric and adult cases, respectively. Inactivation occurs by deletion in 21% and by point mutation in 79% of inactivation cases and correlates with poor survival in adult T-ALL (Van Vlierberghe P et al Nat Gen. 42:338-42) L05559 SH2D1A, ex 1 NM_ ; TGAGCTCGTTTT-AACTGAAGTGTG kb L23888 PHF6, ex 1 NM_ ; TCTTTCTCTTTA-CCCTCATTGGCG 4.6 kb L23843 PHF6, ex 3 NM_ ; CACACTCTGATA-ATGAAAGTCTTG 35.8 kb L23884 PHF6, ex 7 NM_ ; AGCCCTAGTGAC-ACCAGGCCTAAA 12.1 kb -

7 Length (nt) SALSA MLPA probe Gene /Exon Ligation site Partial sequence (24 nt adjacent to ligation site) Distance to next probe L24481 PHF6, ex 10c NM_ ; TTGTTTAGTGGA-TCCATACTCAAA kb L02296 ARHGEF6, ex 19 NM_ ; GATGCTCAAATC-CTTAAAGTGATC - Ж This probe consists of three parts and has two ligation sites. «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. Flanking probe. Included to facilitate determination of the extend of a deletion / duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition being tested. 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 probemix P383 T-ALL Page 7 of 9

8 SALSA MLPA probemix P383-A1 T-ALL sample pictures D ye S ign al Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P383-A1 T-ALL (lot A1-0213) D ye S ign al Size (nt) Figure 2. Capillary electrophoresis pattern from a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P383-A1 T-ALL (lot A1-0213). SALSA probemix P383 T-ALL Page 8 of 9

9 Implemented Changes compared to the previous product description version(s). Version January 2017 (T08) - Warning added in Table 1, 400 nt probe L Various minor textual changes. Version 04 (T06) - Various minor layout changes. Version 03 (T03) - Product description adapted to the updated product version (version number changed, Tables 1 and 2 modified). Version 02 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 01 (46) - Not applicable, new document. SALSA probemix P383 T-ALL Page 9 of 9