Use of inverse PCR to amplify and sequence breakpoints of HPRT deletion and translocation mutations

Abstract

Deletion and translocation mutations have been shown to play a significant role in the genesis of many cancers. The hprt gene located at Xq26 is a frequently used marker gene in human mutational studies. In an attempt to better understand potential mutational mechanisms involved in deletions and translocations, inverse PCR (IPCR) methods to amplify and sequence the breakpoints of hprt mutants classified as translocations and large deletions were developed. IPCR involves the digestion of DNA with a restriction enzyme, circularization of the fragments produced, and PCR amplification around the circle with primers oriented in a direction opposite to that of conventional PCR. The use of this technique allows amplification into an unknown region, in this case through the hprt breakpoint into the unknown joined sequence. Through the use of this procedure, two translocation, one inversion, and two external deletion hprt breakpoint sequences were isolated and sequenced. The isolated IPCR products range in size from 0.4 to 1.8 kb, and were amplified from circles ranging in size from 0.6 to 7.7 kb. We have shown that inverse PCR is useful to sequence translocation and large deletion mutant breakpoints in the hprt gene.

Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology

“…Since repeated regions might be prone to genome rearrangements and therefore prone to HPV integration, we scanned the adjacent regions using the UCSC hg19 genome browser RepeatMasker track for human repeat elements and found a nearby 158 bp long interspersed nuclear element (LINE): L1MB5 located from Chr5 nt position 149,347,143 to 149,347,300. Indeed, L1MB5-derived sequences have been documented as breakpoints, such as in the human genes HPRT [44], CYP2C [45], and in proximity of genes containing the ubiquitin ligase Mib-herc2 domain, which mediates Notch signalling [46]. Strikingly, this domain contains the Hect region, homologous to the E6-associated protein carboxyl terminus, raising the question of whether or not the underlying homology could play a role in this target site selection.…”

Molecular analysis of glycosylphosphatidylinositol anchor deficient aerolysin resistant isolates in gulf war i veterans exposed to depleted uranium

“…Supporting Information Material S7 compiles the 22 sequenced HPRT1 large deletion breakpoints that were found in the literature (Monnat Jr. et al, 1992;Morris and Thacker, 1993;Hou, 1994;Lippert et al, 1997;Steen et al, 1997;Tvrdik et al, 1998;Brooks et al, 2001;Tachibana et al, 2001;Williams et al, 2002;Boone et al, 2010) to the 14 PIGA simple deletion mutants studied here (the possible deletion/translocations were excluded). For the HPRT1 mutants, 10 of the 22 (45%) had two or more repeated bases at the breakpoint and 6 of 22 (27%) had inserted bases.…”

In vitro studies of the genotoxicity of ionizing radiation in human G0 T lymphocytes

“…Although the Southern blot results appear to be consistent with translocation events, direct demonstration by cytogenetic karyotype or translocation breakpoint sequencing would be the definitive evidence [Kodama et al, 1989;Simpson et al, 1993]. One mutation induced by 3 Gy g irradiation in vitro has been shown to be the result of a pericentric inversion involving the HPRT gene at Xq26 and sequences mapped to Xq23 by inverse PCR and fusion point sequencing [Williams et al, 2002]. Three other mutants have been characterized by cytogenetic FISH and banded karyotype and are reported here (Table VI).…”

“…Deletions and rearrangements occur at approximately equal frequency (Tables III and IV), with total gene deletions the most frequent deletion event (22/45) and rearrangements involving the 3 0 portion of the gene, the predominate rearrangement site (15/55). The extent of some of these deletions in terms of breakpoints, flanking markers, and physical size has been determined [Nicklas et al, 1990b;Lippert et al, 1995a,b;Rainville et al, 1995;Williams et al, 2002].…”