RT Journal Article
SR Electronic
T1 Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.02.26.965814
DO 10.1101/2020.02.26.965814
A1 Ahreum Kim
A1 Jong-Yeon Shin
A1 Jeong-Sun Seo
YR 2020
UL http://biorxiv.org/content/early/2020/02/27/2020.02.26.965814.abstract
AB DHFR gene amplification is present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. However, little is known about DHFR gene amplification due to difficulties in quantifying amplification size and recognizing the repetitive rearrangements involved in the process. In this study, we have 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). Amplification of the DHFR gene was optimized to generate homogenously amplified patterns. The amplification units of 11 genes, from the DHFR gene to the ATP6AP1L gene position on chromosome 5 (~2.2Mbp), and a twenty-fold tandemly amplified region were verified using long-range genome and RNA sequencing data. In doing so, a novel inversion 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 might stimulate chromosomal breakage and cause the dysregulation of mismatch repair pathways. Using Hi-C technology, high adjusted interaction frequencies were detected on the amplified unit and unsuspected position on 5q, which could have a complex network of spatial contacts to harbor gene amplification. Characterizing the tandem gene-amplified unit and genomic variants as well as chromosomal interactions on intra-chromosome 5 can be critical in identifying the mechanisms behind genomic rearrangements. These findings may give new insight into the mechanisms underlying the amplification process and evolution of drug resistance.A3SSAlternative 3’ splice siteA5SSAlternative 5’ splice siteBAMBinary alignment mapBFBBreakage-fusion-bridgeCNVCopy number variationDEGDifferentially expressed geneDELDeletionDHFRDihyrofolate reductaseDUPDuplicationFDRFalse discovery rateFISHFluorescent In Situ HybridizationFPKMFragment per kilo base per millionGATKGenome analysis toolkitGSEAGene set enrichment analysisHi-CHigh throughput chromosome conformation captureIGVIntegrative genomics viewerINVInversionINVDUPInverted duplicationKEGGKyoto encyclopedia of genes and genomesLsignSum of the sign of the entries in the lower triangleLvarthe variance of the lower triangleMTXMethotrexateMXEMutually exclusive exonncRNAnon-coding RNANGSNext generation sequencingPacBioPacific BioscienceRIRetained intronRNA-seqRNA sequencingSESkipped exonSMRTSingle molecule real-timeSNVSingle nucleotide variationScoreConner scoreSTARSpliced transcripts alignment to a referenceSVStructural variationTADTopologically associating domainsUsignSum of the sign of the entries in the upper triangleUvarThe variance of the upper triangleVSDVariance stabilizing dataWGSWhole-genome sequencing