Application of Chromosome Conformation Capture Method for Detection MYC/TRD Chromosomal Translocation in Leukemia Cell Line
Background: Chromosomal breakpoints are the most common cause of hereditary diseases and cancers. Today, many standard clinical methods such as cytogenetic and PCR based techniques are used which have limitation regarding detection resolution. Chromosome conformation capture is a method for detecting gene proximity and chromosomal rearrangements.
Materials and Methods: In this study, SKW3 cell line was used for detecting t(8;14)(q24;q11) using a 3C-based technique. SKW3 cell line was used for 3C library preparation. For Inverse PCR, two regions were selected in upstream and downstream of the viewpoint locus on chromosome 8-MYC gene based on EcoRI restriction sites. The captured sequence with intra-chromosomal interaction between chr8-c-MYC and chr14-TRD was selected for the translocation PCR primer design.
Results: The DNA fragment captured in 3C PCR showed a specific TRD sequence translocated downstream of the MYC gene. Translocation PCR demonstrated the existence of (8; 14) (q24; q11) MYC /TRD in both library and genomic DNA.
Conclusion: This result demonstrated 3C- based method could be used as a useful low-cost easy operating technique in chromosomal rearrangements detection. In this study, the integration of whole genome library monitoring and PCR method was used as a high- through put method in chromosomal breakpoints detection.
2. Dong Z, Jiang L, Yang C, et al. A robust approach for blind detection of balanced chromosomal rearrangements with whole‐genome low‐coverage sequencing. Hum Mutat . 2014;35(5):625-36.
3. Roix JJ, McQueen PG, Munson PJ, et al. Spatial proximity of translocation-prone gene loci in human lymphomas. Nat Genet. 2003;34(3):287-91.
4. Zheng J. Oncogenic chromosomal translocations and human cancer. Oncol Rep. 2013;30(5):2011-9.
5. Huntly BJ, Gilliland DG. Leukaemia stem cells and the evolution of cancer-stem-cell research. Nat Rev Cancer. 2005;5(4):311-21.
6. Kubiak M, Lewandowska MA. Can chromatin conformation technologies bring light into human molecular pathology? Acta Biochim Pol. 2015;62(3):483-9
7. Schilit SL, Morton CC. 3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants. Hum Genet. 2018;137(1):55-62.
8. Dekker J, Rippe K, Dekker M, et al. Capturing chromosome conformation. Science. 2002;295(5558):1306-11.
9. Tolhuis B, Palstra R-J, Splinter E, et al. Looping and interaction between hypersensitive sites in the active β-globin locus. Mol Cell. 2002;10(6):1453-65.
10. Williams Jr RL, Starmer J, Mugford JW, et al. fourSig: a method for determining chromosomal interactions in 4C-Seq data. Nucleic Acids Res. 2014; 42(8): e68.
11. Denker A, De Laat W. The second decade of 3C technologies: detailed insights into nuclear organization. Genes Dev. 2016;30(12):1357-82
12. Simonis M, Klous P, Homminga I, et al. High-resolution identification of balanced and complex chromosomal rearrangements by 4C technology. Nat Methods. 2009;6(11):837-42.
13. Homminga I, Pieters R, Langerak AW, et al. Integrated transcript and genome analyses reveal NKX2-1 and MEF2C as potential oncogenes in T cell acute lymphoblastic leukemia. Cancer Cell. 2011;19(4):484-97.
14. Harewood L, Kishore K, Eldridge MD, et al. Hi-C as a tool for precise detection and characterisation of chromosomal rearrangements and copy number variation in human tumours. Genome Biol. 2017; 18(1):125-11.
15. Drexler HG, Gaedicke G, Minowada J. Isoenzyme studies in human leukemia-lymphoma cell lines—I. Carboxylic esterase. Leuk Res. 1985;9(2):209-29.
16. Hirano T, Kishimoto T, Kuritani T, et al. In vitro immune response of human peripheral lymphocytes: IV. Specific induction of human suppressor T cells by an antiserum to the T leukemia cell line HSB. J Immunol. 1979;123(3):1133-40.
17. Ea V, Forné T, Court F. Quantitative Chromosome Conformation Capture (3C-qPCR). Methods Mol Biol . 2017;1589:75-88.
18. Veronese ML, Ohta M, Finan J, Nowell P, Croce CM. Detection of myc translocations in lymphoma cells by fluorescence in situ hybridization with yeast artificial chromosomes. Blood. 1995;85(8):2132-8.
19. Sun LK, Showe LC, Croce CM. Analysis of the 3'flanking region of the human c-myc gene in lymphomas with the t (8; 22) and t (2; 8) chromosomal translocations. Nucleic Acids Res. 1986;14(10):4037-50
20. Kidd JM, Graves T, Newman TL, et al. A human genome structural variation sequencing resource reveals insights into mutational mechanisms. Cell. 2010;143(5):837-47.
21. Wiemels J. Chromosomal translocations in childhood leukemia: natural history, mechanisms, and epidemiology. J Natl Cancer Inst Monogr. 2008;(39):87-90.
22. Kirchhoff M, Rose H, Maahr J, et al. High resolution comparative genomic hybridisation analysis reveals imbalances in dyschromosomal patients with normal or apparently balanced conventional karyotypes. Eur J Hum Genet . 2000 Sep;8(9):661-8.
23. Gribben JG, Neuberg D, Freedman AS, et al. Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma. Blood. 1993;81(12):3449-57.
24. Greaves MF, Wiemels J. Origins of chromosome translocations in childhood leukaemia. Nat Rev Cancer . 2003;3(9):639-49.
25. Lower KM, Hughes JR, De Gobbi M, et al. Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition. Proc Natl Acad Sci U S A. 2009;106(51):21771-6.
26. de Wit E, de Laat W. A decade of 3C technologies: insights into nuclear organization. Genes Dev. 2012;26(1):11-24.
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