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.

DHFR基因扩增通常存在于耐甲氨蝶呤 (MTX) 的结肠癌细胞和急性淋巴细胞白血病中。在这项研究中，我们提出了一个综合框架，通过结合使用单分子实时测序、下一代光学作图和染色体构象捕获 (Hi-C) 来表征扩增区域。我们鉴定了一个跨越 11 个基因的扩增单元，从DHFR基因到ATP6AP1L基因位置，在 5 号染色体上具有高调整的相互作用频率（~2.2 Mbp）和 20 倍串联扩增区域，以及在开始和结束位置的新倒位大多数MSH和MLH中的扩增区域以及移码插入检测到基因。这些突变可能会刺激染色体断裂并导致错配修复的失调。表征串联基因扩增单元可能对于确定触发基因组重排的机制至关重要。这些发现可能为放大过程和耐药性演变的潜在机制提供新的见解。