| Cervical cancer is one of the most common cancers of the womenworldwide, and the infection of the high-risk types of Humanpapillomavirus is considered as the main cause of cervical cancer.The distribution of HPV types, subtypes and the variants variesgreatly worldwide, and is closely related to the corresponding riskof cervical cancer. Therefore, determination of type-specific HPVprevalence and variability of the gene in a region constitutes animportant step towards development of vaccines for prevention ofcervical cancer.In this study, we have investigated cervical HPV prevalence in 51 womencervical cancer tissue in Guangyuan area using combination of 4 differentmethods including PCR using GP5+/6+ consensus primers, E6 type-specificprimers, nested PCR in L1 gene and in HPV 16 E6. In addition, we sequencedthe E6 gene of HPV16, which is predominant HPV type in this area, anddetermined the subtype of the HPV16 based on the sequence of the E6 gene.And also, we compared these four methods for setting more effective method.This work will be the foundation for further large-scale HPV epidemicinvestigation and HPV prevention and development of treatment vaccine inour country.HPV DNA was detected in 49 of the 51 patients with cervical cancer(96.1 percent) and the most common HPV type in patients was type 16,72.5%(35/51) of the patients were infected with this type. HPV type 58 wasthe second most common type, with a prevalence of 11.8 percent (6/51). HPVtype 18, 31, 45, 52, 33, 39 and 59 were each identified in one specimen (1.9percent). Beside of this, there were four specimens we did not classified theirtype. Our result show that the most common types were 16 and 58 inGuangyuan, this result is different from most other region of the world. Theprevalence of type 58 was quite high in this area though the overall prevalenceof only 2.0 percent worldwide.The HPV 16 was the most common in worldwide, and which is found in72.5% of all specimens in our study. Numerous variants of HPV16 have beenidentified in different geographic locations and ethnic groups, and resentinvestigations have evaluated association of specific HPV 16 variants withviral persistence and with the development of cervical cancer. Yamada grouphave carried out analysis on HPV16 samples from 22 different countries, andgeographically mapped its subtypes to different areas of the world. However,there has not any information about prevalence of HPV-16 subtype in Chinayet.In present study, the E6 genes of HPV16 variants were isolated andamplified by PCR from the tumor tissues of patients with cervical cancer inGuanyuan, and the E6 genes were sequenced for investigating their variabilityand the distribution of HPV16 subtypes in this area. 14 different variantsinclude prototype and East Asian type of HPV E6 gene were identified in 33sample analyzed. The type 14 variant known as the prototype gene sequencewas evident in 9 of the 33 samples (27.2%) as the most common HPV16subtype in this area. The 10 variant type known as the East-Asian type genesequence was observed in 2 of the 33 samples (6.1%),which had a substitutionat nt 178(T→G)compared to prototype, and rest 12 variants we found werebelonging to the E-class same as East-Asian type do.There were 12 base substitutions detected in HPV16 E6 ORF region, inthe nucleotide position, 168(C→G), 173(C→T), 176(G→A), 178(T→G), 203(A→G), 240(C→G), 276(A→G), 335(C→T), 350(T→G), 442(A→C), 464(A→G)and 511(T→C)respectively, resultingin 11 change at amino acid, S22T, Y24H, N25D, E25D, Q29E, E34K, G46A,S58N, Y78H, V83L, D113E and C136C. The mutations 203(A→G), 240(C→G)and 511(T→C)have not been reported previously. Our results show thatthe T350G mutation seems to be quite dominant in this region. This mutanthas been reported previously that it can increase the risk for invasive cervicalcancer. 13 samples (39.4%) contained this variant, either alone (6.1%) or incombination with other mutations (33.3). In Yamada's data have shown that,the percentage of T350G variants in European was 44.0%, which were rathersimilar to ours. Besides the mutation C335T+T350G, regarded as moreoncogenic in Indian were found in 4 samples (12.1%) in our data, and was thesecond common subtype in this area.All amino acid mutation we found except D113E were located in theHPV E6 epitope regions, suggests that these mutations may alter their epitopespecificity. The mutation D113E were located in the HPV E6 P53 binding anddegeneration regions;indicate that this mutation may affect the bindingaffinity of E6 to p53.From the phylogenetic tree inferred from the E6 gene of HPV16 variants,we can found that seven variants we found were close to prototype, and fourvariants were close to E131 subtype. Usually, the prototype, East-Asian typeand the E131 type were classified as E-class. What is worth paying attentionis that different between variant 12 and prototype was at 5 nt, while differentbetween variant 12 and Asian-American type was 2 nt, which indicate that thevariant 12 may be the intermediate bridge between the prototype andAsian-American type.In this study, we used four different PCR methods for HPV detection, theGP5+/6+ consensus primers PCR, the E6 type-specific primers PCR, theMY+GP nested PCR and HPV16 E6 nested PCR. Each method has itsadvantage and disadvantage.The PCR method with GP5+/6+ consensus primers was easy to handlethan E6 type-specific primers PCR method, and can detect multiple type ofHPV in each reaction. In this study, the positive rate obtained from thismethod was 84.3%, higher than the E6 type-specific primers PCR method.But this method is not recommended when detecting HPV type, because itneed to combine with other techniques, such as sequencing, hybridize etc.This will increase the cost of examination and make harder to handle.The PCR method with E6 type-specific primers has high-pertinence andsensitivity, it can clearly determine the HPV type instantly. In this study, mostof analysis we used this method. However, this method requires special pair ofprimers for detecting each type of HPV, therefore inevitably increasesworkload, and there are some difficulties on covering all types of HPV.L1 gene nested PCR and HPV 16 E6 nested PCR have high sensitivitycompared to PCR with GP5+/6+ consensus primers and E6 type-specificprimers PCR. Here we used these four methods to carry out identificationanalysis, and find out 96.1% of infection rate. Nevertheless, these two nestedPCR methods are generally not recommended for routine diagnosis of HPVinfection, being more cumbersome and more susceptible to contamination. |
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