Project description:DNA Inverted Repeats as an At-risk Motif for Palindromic Gene Amplificatio defines oncogene amplification that is configured as a series of inverted duplications (palindromic gene amplification). There are several, recurrently amplified oncogenes throughout the human genome. However, it remains unclear whether this recurrent amplification is solely a manifestation of increased fitness resulting from random amplification mechanisms, or if genomic locus-specific amplification mechanism plays a role. In this study, we show that the ERBB2 oncogene at 17q12 is susceptible to palindromic gene amplification in HER2-positive breast tumors. We investigated eight tumors in this study, of which five tumors were HER2-positive, and three tumors were HER2-negative. HER2-status was determined by clinical FISH tests. We applied three genomic approaches to investigate the amplification mechanism: (1) copy number analysis by array-CGH on the Affymetrix SNP6.0 platform (8 files), (2) sequencing of DNA libraries enriched with tumor-derived palindromic DNA (Genome-wide Analysis of Palindrome Formation, GAPF-seq) (8 files) and (3) unbiased whole genome sequencing (WGS) (1 file). These molecular data is made available in the dbGaP. Genomic studies using tumor DNA was approved under the Internal Institutional Review Board at the Cleveland Clinic (IRB07-136: EXEMPT: Chromosome Breakage and DNA Palindrome Formation). Specimens were obtained and methods were carried out under the auspices of IRB 7881 (Evaluation of Genetic and Molecular Markers in Patients with Breast Cancer). All patients consented to allow their cancer specimens to be used by researchers in an anonymized fashion. The consent form indicates that publication will take place without identifiers to protect the identity of any specific individual. We observed significant and enrichment of palindromic DNA within amplified ERBB2 genomic segments in four out of five HER2-positive tumors. None of three HER2-negative tumors showed such enrichment. Palindromic DNA was particularly enriched at amplification peaks and boundaries between amplified and normal copy-number regions. Thus, palindromic gene amplification shaped the amplified ERBB2 locus. The moderate enrichment of palindromic DNA throughout the amplified segments leads us to propose that the ERBB2 locus is amplified through a mechanism that repeatedly generates palindromic DNA, such as Breakage-Fusion-Bridge cycles. Our results reveal a potential interaction between local genomic environments and gene amplification mechanisms. This study is published under the title "Palindromic amplification of the ERBB2 oncogene in primary HER2-positive breast tumors" <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=28211519" target="_blank">(PMID:28211519)</a>.

Project description:DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution.

Project description:DNA replication errors are a major driver of evolutionâ??from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The modelâ??Origin-Dependent Inverted-Repeat Amplification (ODIRA)â??proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication errorâ??the ligation of leading and lagging nascent strands to create a "closed" forkâ??can occur in vitro at short, interrupted inverted repeats. The removal of molecules with closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parentâ??a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homolog prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution These are all CGH arrays comparing DNA copy number between evolved yeast strains and a euploid wt strain.

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DNA Inverted Repeats as an At-risk Motif for Palindromic Gene Amplification

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