Rapid High-Resolution Mapping of Balanced Chromosomal Rearrangements on Tiling CGH Arrays
2011; Elsevier BV; Volume: 13; Issue: 6 Linguagem: Inglês
10.1016/j.jmoldx.2011.07.005
ISSN1943-7811
AutoresHarvey A. Greisman, Noah G. Hoffman, Hye Son Yi,
Tópico(s)Viral-associated cancers and disorders
ResumoThe diagnosis and classification of many cancers depends in part on the identification of large-scale genomic aberrations such as chromosomal deletions, duplications, and balanced translocations. Array-based comparative genomic hybridization (array CGH) can detect chromosomal imbalances on a genome-wide scale but cannot reliably identify balanced chromosomal rearrangements. We describe a simple modification of array CGH that enables simultaneous identification of recurrent balanced rearrangements and genomic imbalances on the same microarray. Using custom tiling oligonucleotide arrays and gene-specific linear amplification primers, translocation CGH (tCGH) maps balanced rearrangements to ∼100-base resolution and facilitates the rapid cloning and sequencing of novel rearrangement breakpoints. As proof of principle, we used tCGH to characterize nine of the most common gene fusions in mature B-cell neoplasms and myeloid leukemias. Because tCGH can be performed in any CGH-capable laboratory and can screen for multiple recurrent translocations and genome-wide imbalances, it should be of broad utility in the diagnosis and classification of various types of lymphomas, leukemias, and solid tumors. The diagnosis and classification of many cancers depends in part on the identification of large-scale genomic aberrations such as chromosomal deletions, duplications, and balanced translocations. Array-based comparative genomic hybridization (array CGH) can detect chromosomal imbalances on a genome-wide scale but cannot reliably identify balanced chromosomal rearrangements. We describe a simple modification of array CGH that enables simultaneous identification of recurrent balanced rearrangements and genomic imbalances on the same microarray. Using custom tiling oligonucleotide arrays and gene-specific linear amplification primers, translocation CGH (tCGH) maps balanced rearrangements to ∼100-base resolution and facilitates the rapid cloning and sequencing of novel rearrangement breakpoints. As proof of principle, we used tCGH to characterize nine of the most common gene fusions in mature B-cell neoplasms and myeloid leukemias. Because tCGH can be performed in any CGH-capable laboratory and can screen for multiple recurrent translocations and genome-wide imbalances, it should be of broad utility in the diagnosis and classification of various types of lymphomas, leukemias, and solid tumors. Recurrent balanced chromosomal rearrangements, such as translocations and inversions, result in oncogenic fusion genes that participate in the pathogenesis and classification of many cancers.1Mitelman F. Johansson B. Mertens F. 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Oligonucleotide probes representing unmasked genomic segments at high density were designed using a Python algorithm (Tile.py; code available on request) that simultaneously optimizes probe length, distribution, and melting temperature (Tm) as predicted by the dan tool (EMBOSS version 3.0.0) (http://emboss.sourceforge.net/apps/cvs/emboss/apps/dan.html) using default parameters. Individual probes (maximum length, 60 nt) were shortened incrementally to achieve an optimal Tm of 74.5°C (minimum length, 25 nt). Tile.py parameters were selected to yield a median spacing of ∼60 nt between adjacent probes in unmasked regions. Characteristics of the lymphoma and myeloid leukemia array probes are summarized in Supplemental Tables S1 and S2 (available at http://jmd.amjpathol.org). The array designs are accessible in the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) (http://www.ncbi.nlm.nih.gov/geo) under platform accession numbers GPL10863 (lymphoma array) and GPL10864 (leukemia array). Primers previously optimized for PCR amplification generally performed well in tCGH experiments. VDJ-type translocation breakpoints on the der(14), for example, were amplified using the BIOMED-2 JH consensus primer44van Dongen J.J. Langerak A.W. Brüggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H. Droese J. González D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2497) Google Scholar and reciprocal VDJ-type breakpoints were amplified using an equimolar mixture of the seven BIOMED-2 DH primers (Table 1). For the design and selection of linear amplification primers for switch region (SH) breakpoints, see Supplemental Figure S1 (available at http://jmd.amjpathol.org). Primers targeting unique sequences were selected using the online tool Primer3 version 0.4.0 (http://frodo.wi.mit.edu/primer3) and default Primer3 settings, except that the optimum primer length was 25 nt, the optimum Tm was 65°C, and the human mispriming (repeat) library was used. Primers within interspersed repeats or that overlapped single nucleotide polymorphisms, copy number variations, or other repetitive motifs were avoided whenever possible.Table 1Linear Amplification PrimersPrimerSequenceGenome position (NCBI36/hg18)JH consensus⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-5CTTACCTGAGGAGACGGTGACC-3′See textDH1⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-GGCGGAATGTGTGCAGGC-3′See textDH2⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-GCACTGGGCTCAGAGTCCTCT-3′See textDH3⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-GTGGCCCTGGGAATATAAAA-3′See textDH4⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-AGATCCCCAGGACGCAGCA-3′See textDH5⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-CAGGGGGACACTGTGCATGT-3′See textDH6⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-TGACCCCAGCAAGGGAAGG-3′See textDH7⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-CACAGGCCCCCTACCAGC-3′See textSpF†Y = C or T; R = A or G.5′-GCYCAGCYCAGCYCA-3′See textSpR†Y = C or T; R = A or G.5′-GRGCTGRGCTGRGCT-3′See textSγF5′-GGCTGCTCTGCCCTGGTCCCCTGAGCTCCA-3′See textSγR5′-TGGAGCTCAGGGGACCAGGGCAGAGCAGCC-3′See textMYC-F5′-GGTCGGACATTCCTGCTTTA-3′chr8:128,818,341–128,818,360BCL6-R⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-AGAATTCCAGAGGCCGAGCTTTGCTACAGCGAAGG-3′chr3:188,946,258–188,946,292PML-1F5′-TGCTGCCTAGTCATTTCTGACTCAA-3′chr15:72,111,924–72,111,948PML-2F5′-AACATGCCATGATTCAAAGTCTGGT-3′chr15:72,102,110–72,102,134BCR-1F5′-AGGCTCATCATTCTCACCTATGCAG-3′chr22:21,961,112–21,961,136BCR-2F5′-CCAGACCAGCACTGCACTTGAGAG-3′chr22:21,961,533–21,961,556MLL-1F⁎JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44), primer BCL6/41-R is from Akasaka et al45, and primer MLL-P1LR-F is from Langer et al.465′-CCGCCTCAGCCACCTACTACAGGACCG-3′chr11:117,857,921–117,857,947MLL-2F5′-CATGTTCTAGCCTAGGAATCTGCTT-3′chr11:117,859,537–117,859,561MLL-3F5′-TTATAGCACCAGTCCTTCAACTTCTG-3′chr11:117,863,159–117,863,184MYH11-1F5′-GAAGTTTCCACACCAACCATGAGAG-3′chr16:15,722,110–15,722,134MYH11-2F5′-CAGTGTCAATGACTGAATCCAGGTG-3′chr16:15,725,035–15,725,059MYH11-3F5′-AGGGTAAATGGCTATGCCAAGTGAA-3′chr16:15,727,132–15,727,156MYH11-4F5′-AGGTGTCGTGTGATTGACACTGCTA-3′chr16:15,729,863–15,729,887MYH11-5F5′-CCGAAAGTGTTGTTACACCTTTGCT-3′chr16:15,732,551–15,732,575RUNX1-1R5′-CAACAGATATGTTCAGGCCACCAAC-3′chr21:35,153,781–35,153,805RUNX1-2R5′-TCTGGATGAATAAGACCTCCCGAGT-3′chr21:35,150,980–35,151,004RUNX1-3R5′-GCATGTAAACTAAGGCGTCCAGATG-3′chr21:35,147,666–35,147,690RUNX1-4R5′-GAAACACTCTGGGCACTGTTCCTAA-3′chr21:35,144,843–35,144,867RUNX1-5R5′-AACCATGTTGCCCTACTTCCTTGAG-3′chr21:35,141,919–35,141,943RUNX1-6R5′-TGCCCATTATGTATGAGATCTGCTG-3′chr21:35,138,636–35,138,660RUNX1-7R5′-TGAGTGCTTGCTTTCCTGTTACCAC-3′chr21:35,135,571–35,135,595RUNX1-8R5′-GCAGCTCGGTTATCAACGAGATATG-3′chr21:35,132,335–35,132,359RUNX1-9R5′-GTTGTGCAATCGATCAAGGACTCTT-3′chr21:35,129,871–35,129,895 JH consensus and DH primers are from the BIOMED-2 consensus (van Dongen et al44van Dongen J.J. Langerak A.W. Brüggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H. Droese J. González D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2497) Google Scholar), primer BCL6/41-R is from Akasaka et al45Akasaka H. Akasaka T. Kurata M. Ueda C. Shimizu A. Uchiyama T. Ohno H. Molecular anatomy of BCL6 translocations revealed by long-distance polymerase chain reaction-based assays.Cancer Res. 2000; 60: 2335-2341PubMed Google Scholar, and primer MLL-P1LR-F is from Langer et al.46Langer T. Metzler M. Reinhardt D. Viehmann S. Borkhardt A. Reichel M. Stanulla M. Schrappe M. Creutzig U. Ritter J. Leis T. Jacobs U. Harbott J. Beck J.D. Rascher W. Repp R. Analysis of t(9;11) chromosomal breakpoint sequences in childhood acute leukemia: almost identical MLL breakpoints in therapy-related AML after treatment without etoposides.Genes Chromosomes Cancer. 2003; 36: 393-401Crossref PubMed Scopus (68) Google Scholar† Y = C or T; R = A or G. Open table in a new tab To amplify across large intronic breakpoint regions in myeloid fusion partners such as MYH11 and RUNX1, we designed tandem arrays of linear amplification primers (Table 1) distributed at 2- to 3-kb intervals across the region of interest, which typically comprises one or more contiguous introns and is bounded by the flanking exons. Briefly, the first primer at the 5′ end of the array was selected using the Primer3 online tool and the criteria given above to search a 0.5- to 1.5-kb genomic segment located just upstream of the 5′ flankin
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