Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations in mismatch repair genes in methotrexate-resistant HT-29 cells

DHFR gene amplification is commonly present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. In this study, we proposed an integrative framework to characterize the amplified region by using a combination of single-molecule real-time sequencing, next-generation optical mapping, and chromosome conformation capture (Hi-C). We identified an amplification unit spanning 11 genes, from the DHFR gene to the ATP6AP1L gene position, with high adjusted interaction frequencies on chromosome 5 (~2.2 Mbp) and a twenty-fold tandemly amplified region, and novel inversions at the start and end positions of the amplified region as well as frameshift insertions in most of the MSH and MLH genes were detected. These mutations might stimulate chromosomal breakage and cause the dysregulation of mismatch repair. Characterizing the tandem gene-amplified unit may be critical for identifying the mechanisms that trigger genomic rearrangements. These findings may provide new insight into the mechanisms underlying the amplification process and the evolution of drug resistance. Cancer chemotherapy: Sequencing surplus gene copies Sequencing a large region of DNA containing many surplus copies of genes linked to drug resistance in colon cancer cells may illuminate how these genomic rearrangements arise. Such regions of gene amplification are highly repetitive, making them impossible to sequence using ordinary methods, and little is known about how they are generated. Using advanced methods, Jeong-Sun Seo at Seoul National University Bundang Hospital in South Korea and co-workers sequenced a region of gene amplification in colon cancer cells. The amplified region was approximately 20 times the length of that in healthy cells and contained many copies of an eleven-gene segment, including a gene implicated in drug resistance. The region also contained mutations in chromosomal repair genes which would disrupt repair pathways. These results illuminate the genetic changes that lead to gene amplification and drug resistance in cancer cells.