Breakpoint Mapping And Its Significance On Large Deletions Of Chromosome9p21

Background and AimsCancer is one main public health problem in the whole world. The global annual incidence of cancer is about202.8/100,000while its global annual morbility is about127.9/100,000. In the United States, one of every four deaths is caused by cancer. Tumor incidence grows gradually every year in China, which has already been the first cause of death. In the early20th centry, scientists had found that chromosomal structural variations are key factors of cancer initiation and development. The Philadelphia Chromosome further confirms that chromosomal rearrangements can result in epigenetic alterations and promote the initiation and development of cancers. Copy number variations, mainly including deletions and amplifications, are main parts of chromosomal changes. Copy number variations detected based on microarrays can not be charaterized in base pair level. In order to analyze possible epigenetic alterations caused by copy number variations, it is necessary to modify useful methods for breakpoint mapping. Focused on large deletions on chromosome9p21, this study is aimed to find out efficient methods to detect breakpoints as well as to analyze the possible changes on transcription level in breast cancer cell line MCF-7and pancreatic cancer cell line PANC-1.MethodsCell line DNA and RNA were extracted from cultured breast cancer cell line MCF-7and pancreatic cancer cell line PANC-1. MLPA (Multiplex Ligation-based Probe Amplification) was used to detect large deletions on chromosome9p21in these two cell lines, with deletion borders furtherly confirmed by conventional PCR. The long range and inverse PCR were used to detect deletion breakpoints on chromosome9p21in these two cell lines. RACE (Rapid Amplification of cDNA ends), TA cloning and sequencing were used to find out possible new transcripts.Results1,The large deletions on chromosome9p21, with invovlment in MTAP and CDKN2A genes, were found in both MCF-7and PANC-1cell lines. The homozygous deletions in MCF-7was found starting within MTAP gene and ending in CDKN2A gene while the homozygous deletions in PANC-1was found starting within MTAP gene, with the centromeric border beyond the detection range of MLPA method.2,The long range and inverse PCR confirmed the deletions spanning from chr9:21819532to chr9:21989622in MCF-7, with a deletion size of170kb, starting within the Intron4of MTAP on the tolemeric side and ending within the Intron1near Exon1β of CDKN2A on the centromeric side.3,In breast cancer cell line MCF-7, A new MTAP fusion transcript identified turned out to be fused between the first4exons of MTAP and non-coding region chr9:22087143-22087648, containing967base pairs on cDNA level and translating into a protein of125amino acids. In pancreatic cancer cell line PANC-1, two new MTAP fusion transcripts were identified. One transcript turned out to be fused between the first4exons of MTAP and non-coding region chr9:22384168-22384481, containing867base pairs on cDNA level and translationg into one protein of138amino acids. The other one turned out to be fused between the first exons of MTAP and non-coding region chr9:22674440-22674564, containing598base pairs on cDNA level and translating into149amino acids.ConclusionsThe MLPA method is an effient way to detect large deletions correctly, which is suitable to confirm abnormal regions detected by array-based Comparative Genomic Hybridization. Both the breast cancer cell line MCF-7and pancreatic cancer cell line PANC-1contain large deletions involved in MTAP and CDKN2A on chromosome9p21. The long range/inverse PCR confirmed the large deletion in MCF-7spanning from chr9:21819532to chr9:J21989622, with a deletion size of170kb, starting within the Intron4of MTAP and ending within the Intron1near Exon1β of CDKN2A. Due to large deletions on chromosome9p21, one new MTAP fusion transcript and two new MTAP fusion transcripts were found in MCF-7, PANC-1, respectively.It needs further study whether these new MTAP fusion transcripts are involved in the initation and development of breast/pancreatic cancer or not.