Xie Huaping, Rachakonda P Sivaramakrishna, Heidenreich Barbara, Nagore Eduardo, Sucker Antje, Hemminki Kari, Schadendorf Dirk, Kumar Rajiv
Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Division of Molecular Genetic Epidemiology, German Cancer Research Center, Heidelberg, Germany.
Oncotarget. 2016 Mar 29;7(13):16490-504. doi: 10.18632/oncotarget.7503.
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.
位于9号染色体p21区域、包含CDKN2A和CDKN2B肿瘤抑制基因的基因组位点，在多种人类癌症中通过突变、缺失和启动子甲基化而失活。此外，该位点编码一种反义RNA（ANRIL）。该位点靶向多个基因的半合子和纯合子缺失在不同癌症中相当常见。在本研究中，我们使用阵列比较基因组杂交（aCGH）技术，对来自转移性肿瘤、先前已鉴定出纯合子缺失的5种黑色素瘤细胞系中的断点进行了研究。对于断点定位，我们使用了引物近似多重PCR（PAMP）和反向PCR技术。我们的结果显示，3种细胞系存在复杂重排。在另外2种细胞系中，分别有141 kb和181 kb的局灶性缺失，我们鉴定出了涉及MTAP和ANRIL的融合基因产物。我们还在对应于3种细胞系的肿瘤组织DNA中证实了复杂重排和局灶性缺失。对转录本进行的3' cDNA末端快速扩增（3' RACE）鉴定出了MTAP-ANRIL融合基因的3种异构体。对64种黑色素瘤细胞系的cDNA进行筛查，结果在13种（20%）细胞系中检测到了融合转录本，这些融合转录本涉及MTAP的外显子4-7和ANRIL基因的外显子2或5。我们还在7种原发性黑色素瘤肿瘤中的2种中检测到了涉及MTAP和ANRIL的融合转录本，这2种肿瘤在该位点存在局灶性缺失。该研究结果除了鉴定出涉及CDKN2A位点的复杂重排外，还显示涉及MTAP和ANRIL基因的融合转录本频繁出现。
