Mapping of deletion breakpoints at the CDKN2A locus in melanoma: detection of MTAP-ANRIL fusion transcripts
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Huaping Xie1,2, P. Sivaramakrishna Rachakonda2, Barbara Heidenreich2, Eduardo Nagore3, Antje Sucker4, Kari Hemminki2,5, Dirk Schadendorf4,6, Rajiv Kumar2
1Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
2Division of Molecular Genetic Epidemiology, German Cancer Research Center, Heidelberg, Germany
3Department of Dermatology, Instituto Valenciano de Oncologia, Valencia, Spain
4Department of Dermatology, University Hospital Essen, Essen, Germany
5Center for Primary Health Care Research, Lund University, Malmö, Sweden
6German Cancer Consortium (DKTK), Essen, Germany
Rajiv Kumar, e-mail: email@example.com
Keywords: melanoma, CDKN2A, deletions, break points
Received: December 28, 2015 Accepted: February 11, 2016 Published: February 19, 2016
Genomic locus at chromosome 9p21 that contains the CDKN2A and CDKN2B tumor suppressor genes is inactivated through mutations, deletions and promoter methylation in multiple human cancers. Additionally, the locus encodes an anti-sense RNA (ANRIL). Both hemizygous and homozygous deletions at the locus targeting multiple genes are fairly common in different cancers. We in this study investigated breakpoints in five melanoma cell lines, derived from metastasized tumors, with previously identified homozygous deletions using array comparative genomic hybridization (aCGH). For breakpoint mapping, we used primer approximation multiplex PCR (PAMP) and inverse PCR techniques. Our results showed that three cell lines carried complex rearrangements. In two other cell lines, with focal deletions of 141 kb and 181 kb, we identified fusion gene products, involving MTAP and ANRIL. We also confirmed the complex rearrangements and focal deletions in DNA from tumor tissues corresponding to three cell lines. The rapid amplification of 3’cDNA ends (3’RACE) carried out on transcripts resulted in identification of three isoforms of MTAP-ANRIL fusion gene. Screening of cDNA from 64 melanoma cell lines resulted in detection of fusion transcripts in 13 (20%) cell lines that involved exons 4-7 of the MTAP and exon 2 or 5 of the ANRIL genes. We also detected fusion transcripts involving MTAP and ANRIL in two of the seven primary melanoma tumors with focal deletion at the locus. The results from the study, besides identifying complex rearrangements involving CDKN2A locus, show frequent occurrence of fusion transcripts involving MTAP and ANRIL genes.
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