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Updated: Mar 25, 2026
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Mapping of deletion breakpoints at the CDKN2A locus in melanoma: detection of MTAP-ANRIL fusion transcripts
Huaping Xie1,2, P Sivaramakrishna Rachakonda2, Barbara Heidenreich2
1Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Abstract:
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.
Insights
The 9p21 tumor suppressor locus, CDKN2A/B, is frequently altered in cancers. This study found common MTAP-ANRIL fusion transcripts in melanoma, suggesting a role in cancer development.
Area of Science:
- Cancer Genomics
- Molecular Oncology
- Melanoma Research
Background:
- The 9p21 genomic locus, housing CDKN2A and CDKN2B tumor suppressor genes, is frequently inactivated in various human cancers.
- This locus also encodes the anti-sense RNA (ANRIL), and deletions targeting multiple genes within this region are common.
- Understanding genomic alterations at 9p21 is crucial for cancer research.
Purpose of the Study:
- To investigate breakpoints and genomic rearrangements within the 9p21 locus in melanoma cell lines with homozygous deletions.
- To identify fusion gene products resulting from deletions at this locus.
- To assess the frequency of MTAP-ANRIL fusion transcripts in melanoma.
Main Methods:
- Array comparative genomic hybridization (aCGH) for identifying homozygous deletions.
- Primer approximation multiplex PCR (PAMP) and inverse PCR for breakpoint mapping.
- Rapid amplification of 3'cDNA ends (3'RACE) for transcript analysis.
- cDNA screening of melanoma cell lines and primary tumors.
Main Results:
- Complex genomic rearrangements were identified in three melanoma cell lines.
- Focal deletions in two cell lines led to MTAP-ANRIL fusion gene products.
- MTAP-ANRIL fusion transcripts were detected in 20% of melanoma cell lines and in primary melanoma tumors with focal deletions.
Conclusions:
- The study identified complex rearrangements and focal deletions at the CDKN2A locus in melanoma.
- Frequent occurrence of MTAP-ANRIL fusion transcripts was observed in melanoma, indicating their potential significance.
- These findings contribute to understanding the genomic landscape of melanoma.
