Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas
Identifieur interne : 000120 ( Main/Merge ); précédent : 000119; suivant : 000121Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas
Auteurs : Bo Xu [Allemagne] ; Sasithorn Chotewutmontri [Allemagne] ; Stephan Wolf [Allemagne] ; Ursula Klos [Allemagne] ; Martina Schmitz [Allemagne] ; Matthias Dürst [Allemagne] ; Elisabeth Schwarz [Allemagne]Source :
- PLoS ONE [ 1932-6203 ] ; 2013.
Abstract
Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3′-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3′-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.
Url:
DOI: 10.1371/journal.pone.0066693
PubMed: 23824673
PubMed Central: 3688939
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when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3′-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3′-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Schiffman, M" uniqKey="Schiffman M">M Schiffman</name></author><author><name sortKey="Castle, Pe" uniqKey="Castle P">PE Castle</name></author><author><name sortKey="Jeronimo, J" uniqKey="Jeronimo J">J Jeronimo</name></author><author><name sortKey="Rodriguez, Ac" uniqKey="Rodriguez A">AC Rodriguez</name></author><author><name sortKey="Wacholder, S" uniqKey="Wacholder S">S Wacholder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Arbyn, M" uniqKey="Arbyn M">M Arbyn</name></author><author><name sortKey="Castellsague, X" uniqKey="Castellsague X">X Castellsague</name></author><author><name sortKey="De 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