Detection and screening of chromosomal rearrangements in uterine leiomyomas by long‐distance inverse PCR

Abstract

Genome instability is a hallmark of many tumors and recently, next-generation sequencing methods have enabled analyses of tumor genomes at an unprecedented level. Studying rearrangement-prone chromosomal regions (putative "breakpoint hotspots") in detail, however, necessitates molecular assays that can detect de novo DNA fusions arising from these hotspots. Here we demonstrate the utility of a long-distance inverse PCR-based method for the detection and screening of de novo DNA rearrangements in uterine leiomyomas, one of the most common types of human neoplasm. This assay allows in principle any genomic region suspected of instability to be queried for DNA rearrangements originating there. No prior knowledge of the identity of the fusion partner chromosome is needed. We used this method to screen uterine leiomyomas for rearrangements at genomic locations known to be rearrangement-prone in this tumor type: upstream HMGA2 and within RAD51B. We identified a novel DNA rearrangement upstream of HMGA2 that had gone undetected in an earlier whole-genome sequencing study. In more than 30 additional uterine leiomyoma samples, not analyzed by whole-genome sequencing previously, no rearrangements were observed within the 1,107 bp and 1,996 bp assayed in the RAD51B and HMGA2 rearrangement hotspots. Our findings show that long-distance inverse PCR is a robust, sensitive, and cost-effective method for the detection and screening of DNA rearrangements from solid tumors that should be useful for many diagnostic applications.

Detection of subclonal L1 transductions in colorectal cancer by long-distance inverse-PCR and Nanopore sequencing

“…1.25 ng circular templates generated by restriction enzyme digestion and T4 ligation were used in LDI-PCR as eight replicates (Supplementary Fig. S5 ) as previously described 20 using three primer pairs (Supplementary Table S1 ). These PCR products were then analysed on a 1% agarose gel and purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).…”

“…Thus we have developed a direct molecular approach to detect 3′ transductions from specific L1s and hence monitor their activity; this method requires no prior knowledge of the insertion target regions. We apply long-distance inverse (LDI)-PCR 20 to a particular source L1 ( TTC28 specific L1 in this study) by targeting inverse primers to its frequently transduced 3′ flanking sequence (here referred to as the “unique tag”) (Fig. 1b ).…”

Riemerella anatipestifer M949_1360 Gene Functions on the Lipopolysaccharide Biosynthesis and Bacterial Virulence

“…The site of transposon insertion in the mutant strain was identified by inverse polymerase chain reaction (PCR) [ 27 ]. In brief, genomic DNA of the mutant strain was digested with restriction enzyme Hind III at 37°C for 3 h. After purification, the DNA fragment was ligated using T4 DNA Ligase [TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China].…”

A View on Uterine Leiomyoma Genesis through the Prism of Genetic, Epigenetic and Cellular Heterogeneity

“…In ULs, most 12q14-15 breaks are located upstream of the promoter sequence of the HMGA2 gene, upregulating its expression [ 146 , 150 , 151 ]. Along with transcriptional alterations, the higher levels of HMGA2 protein in ULs could be associated with the loss of let-7 regulatory microRNA binding sites in truncated or chimeric transcripts of the damaged HMGA2 gene [ 152 , 153 ].…”