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Journal of Clinical Microbiology, November 1999, p. 3514-3517, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Analysis by Multiplex PCR of the Physical Status of Human Papillomavirus Type 16 DNA in Cervical Cancers

Mitsuo Yoshinouchi,1 Atsushi Hongo,1 Keichiro Nakamura,1 Junichi Kodama,1,* Sachio Itoh,2 Hiroyuki Sakai,3 and Takafumi Kudo1

Department of Obstetrics and Gynecology1 and Department of Molecular Genetics, Institute of Cellular and Molecular Biology,2 Okayama University Medical School, Okayama, and Institute for Virus Research, Kyoto University, Kyoto,3 Japan

Received 22 February 1999/Returned for modification 4 June 1999/Accepted 22 July 1999

Integration of human papillomavirus (HPV) DNA occurs early in cancer development and is an important event in malignant transformation of cervical cancer. Integration of HPVs preferentially disrupts or deletes the E2 open reading frame, which results in the loss of its expression. The preferential disruption of the E2 gene causes the absence of the E2 gene sequences in the PCR product following integration. Twenty-two carcinomas positive for HPV type 16 (HPV-16) DNA were first tested for the disruption of the E2 gene by PCR. A specific fragment of the E2 gene was not amplified in 10 cases, suggesting integration of HPV DNA into the host genome. Next, multiplex PCR for the HPV E2 and E6 genes was carried out in the remaining 12 cases. Copy numbers of both genes should be equivalent in episomal forms, while the E2 gene copy number will be smaller than that for E6 following the preferential disruption of the E2 gene in concominant forms. Although relative ratios of HPV E2 to E6 PCR products (E2/E6 ratios) ranged from 1.40 to 2.34 in 10 of 12 cases, multiplex PCR products from 2 cases displayed extremely low ratios of 0.69 and 0.61. Southern blot hybridization with an HPV-16 probe revealed that only in these two cases was both episomal and integrated HPV DNA being carried simultaneously. Thus, multiplex PCR for the E2 and E6 genes of HPV-16 DNA following PCR for the E2 gene can distinguish the pure episomal form from a mixed form of episomal and integrated HPV DNA. Clinical application of this technique will help researchers to understand the implication of the integration of HPV DNA for cervical carcinogenesis and cervical cancer progression.

* Corresponding author. Mailing address: Department of Obstetrics and Gynecology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan. Phone: (81) 86 235 7320. Fax: (81) 86 225 9570. E-mail: kodama{at}cc.okayama-u.ac.jp.

Journal of Clinical Microbiology, November 1999, p. 3514-3517, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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