| Objectives:To investigate human papillomavirus(HPV) subtypes, HPV16’s E2, E6 and E7 gene mutations and E6, E7 antigen expressions in HPV-related diseases, as well as its relationship to the patients’ clinical features. Methods:1. Ninety-one formalin-fixed paraffin-embedded biopsied tissue stock of HPV-related diseases including 31 condyloma acuminatum(CA), 11 cervical intraepithelial neoplasia grade 2(CIN2), 23 cervical intraepithelial neoplasia grade 3(CIN3), 9 Bowenoid papulosis(BP), 3 Bowen’s disease(BD) and 14 Paget’s disease(PD) in Department of Pathology, Zhongshan Hospital of Xiamen University from 2007 to 2013 were collected.2. HPV genomic DNAs were extracted, and then the HPV subtypes were tested with a PCR- membrane in situ hybridization(PCR-ISH) assay.3. The HPV16 E2, E6, and E7 gene mutations were assayed by DNA sequencing and comparing with published wild-type HPV16 sequences in NCBI with a BLAST software.4. The HPV16 oncogenic E6 and E7 antigens were immunohistochemically stained by streptoavidin peroxidase(S-P) method with antibodies against HPV16 E6 and E7.5. The relationship of HPV subtypes, HPV16 E2, E6, E7 gene mutations the, E6 and E7 antigen expressions, with patients’ clinical features(gender, age, location, and histopathological features) was analyzed.6. The differences of HPV DNA detection rates among different groups were analyzed with Chi square test, in which p<0.05 was set as statistical significance level of difference. Results:1. Ten HPV subtypes were detected in these 91 HPV-related diseases specimens. HR-HPV DNAs were detected in 14 of 31 CA(45.2%), 5 of 11 CIN2(45.5%), 14 of 23 CIN3(60.7%) and 1 of 9 BD(11.1%). Among which, high risk HPV16 DNAs were detected in 3 of 31 CA(9.7%), 2 of 11 CIN2(18.2%), 8 of 23 CIN3(34.8%), 1 of 9 BD(11.1%), but in none of 14 PD or 3 BP. HPV18 DNAs were not detected in any of these 91 specimens. Only LR-HPVs were detected in male patient with CA, whereas both LR-HPVs and HR-HPVs were detected in female patients with CA. In female patients with CA, HR-HPVs were major detected in cervix, and the detection rate in patients aged above 50 were a little bit higher than those aged beyond 50, but the difference had no statistical significance. The detection rares of HPV, HR-HPV, and HPV16 DNAs in CIN3 were a little bit higher than those in CIN2, but the difference was no statistically significant.2. Substitution mutations of HPV16 E2 gene were detected in 1 CA and 1 CIN3, Substitution mutations of HPV16 E2, E6 and E7 genes were detected in 2 CIN3. Ten substitution sites were detected in HPV16 E2 gene, among which G2827A(Aspâ†'Asn), C3158A(Thrâ†'Lys), G3248A(Argâ†'Gln), T3383C(Ileâ†'Thr), C3409T(Proâ†'Ser), G3448A(Gluâ†'Lys), C3683A(Thrâ†'Lys) and C3786A(Aspâ†'Gln) were missense mutations. Five substitution sites were detected in HPV16 E6 gene, among which the G94 A substitution mutation was located upstream of the start codon resulting in change of a regulatory amino acid, G132T(Argâ†'Ile), T178A(Aspâ†'Glu), T178G(Aspâ†'Glu) and C278G(Proâ†'Ala) were missense mutation, the substitution mutations of T178A(Aspâ†'Glu) and T178G(Aspâ†'Glu) encode the same amino acid. Four substitution mutations were detected in HPV16 E7 gene, among which A647G(Asnâ†'Ser) was missense mutation.3. HPV16 E6 and E7 antigen expressions:Among 14 HPV16 DNA positive specimens, 1 BD, 1 CIN2 and 3 CIN3 were positive for HPV16 E6 antigen and 1 BD and 3 CIN3 were positive for HPV16 E7 antigen. Conclusion:1. There were combined infections of LR-HPVs and/or HR-HPVs in female patients with CA, in which the HR-HPVs were major detected on cervix.2. The major subtype of HR-HPV infected in CIN was HPV16.3. There is HPV16 E2 gene substitution mutations in CA.4. Substitution mutations of HPV16 E2, E6 and E7 genes might play a role in the development and progress of CA, CIN2 and CIN3.5. HPV16 E7 antigen expressed only in patients with precancerous or carcinoma in situ, which might thus serve as a predicting factor for malignancy.6. There seemed no relationship of PD to HPV subtypes tested. Keywords:... |
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