Bavin, Peggy Jane; (1992) Human papillomaviruses and their association with cervical disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The overall aim of the project was to establish whether human papillomaviruses (HPV) are associated with cervical intraepithelial neoplasia (CIN) and cervical carcinoma and examine the use of HPV 16 as an indicator of cervical disease. For this purpose, a Southern blot hybridization system was developed to detect HPV DNA in cervical scrape samples. However, following the description of the polymerase chain reaction for detection of nucleic acid a PCR system for detection of HPV DNA was developed. The ability of Southern blot hybridization and PCR to detect HPV DNA in cervical scrape samples was compared to determine the most suitable method for use as a diagnostic test for HPV. The PCR method was 100,000 times more sensitive than Southern blot and was more accurate in identifying women with cervical disease. The PCR system was used to analyse cervical scrape samples from two study groups for the presence of HPV 16 DNA. The first group (Study A) consisted of 200 women from a General Practice population who were expected to have normal cervical cytology.HPV 16 was present in 17% of women with no cervical abnormalities. In those women from Study A who had cervical disease (n = 22) the prevalence of HPV 16 increased with greater severity of disease from 15.4% in those with CIN 1, 40% in those with CIN 2, to 75% of those with CIN 3. The presence of HPV 16 DNA was significantly associated with CIN 2 and 3 (p = 0.009) and was therefore useful as an indicator of severe cervical disease in this population. The ability of PCR for HPV 16 to identify women with disease was compared with that of standard cytological analysis. There was no significant difference between the two methods, although a combination of screening by cytology and PCR resulted in the identification of a higher proportion of women with disease and PCR was associated with a higher false positive rate. The second group (Study B) consisted of 200 women who had been referred to the Royal Free Hospital colposcopy clinic with a smear report suggesting mild dyskaryosis. Within this group there were 54 women who were cytologically normal, 59 women who had CIN 1 or WVI and 66 women with severe cervical disease (CIN 2 or 3). The results of Study B concurred with Study A in demonstrating an increasing prevalence of HPV 16 with greater severity of disease from 53% in women with CIN 1, 64% of women with CIN 2 to 74% of patients with CIN 3. However, the prevalence of HPV 16 in the normal women in Study B was 63%, and this high value precludes the use of HPV 16 as an indicator of severe cervical disease in this population. Duplicate analysis of each cervical scrape sample from Study A and Study B allowed the reproducibility of the HPV 16 PCR system to be determined. The false positive rate was 0.1% and the false negative rate was 0.77%. The long control region (LCR) of HPV 16 was cloned from a woman without cervical disease (CO) and a woman with CIN 3 (C3). The DNA sequence of each isolate was determined and compared with the prototype HPV 16 sequence. Nucleotide variations were evident in both isolates, but LCRC3 shared less homology with the prototype sequence than LCRCO. A single nucleotide mutation occurred within the glucocorticoid responsive element of LCRC3, which disrupts the palindrome of the protein binding domain. The level of expression from the HPV 16 LCR was determined using a chloramphenicol acetyltransferase assay and found to be 5-fold lower than that of the SV40 early promoter.

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