Deep sequencing of HPV E6/E7 genes reveals loss of genotypic diversity and gain of clonal dominance in high-grade intraepithelial lesions of the cervix.

BACKGROUND: Human papillomavirus (HPV) is the carcinogen of almost all invasive cervical cancer and a major cause of oral and other anogenital malignancies. HPV genotyping by dideoxy (Sanger) sequencing is currently the reference method of choice for clinical diagnostics. However, for samples with multiple HPV infections, genotype identification is singular and occasionally imprecise or indeterminable due to overlapping chromatograms. Our aim was to explore and compare HPV metagenomes in abnormal cervical cytology by deep sequencing for correlation with disease states. RESULTS: Low- and high-grade intraepithelial lesion (LSIL and HSIL) cytology samples were DNA extracted for PCR-amplification of the HPV E6/E7 genes. HPV+ samples were sequenced by dideoxy and deep methods. Deep sequencing revealed ~60% of all samples (n = 72) were multi-HPV infected. Among LSIL samples (n = 43), 27 different genotypes were found. The 3 dominant (most abundant) genotypes were: HPV-39, 11/43 (26%); -16, 9/43 (21%); and -35, 4/43 (9%). Among HSIL (n = 29), 17 HPV genotypes were identified; the 3 dominant genotypes were: HPV-16, 21/29 (72%); -35, 4/29 (14%); and -39, 3/29 (10%). Phylogenetically, type-specific E6/E7 genetic distances correlated with carcinogenic potential. Species diversity analysis between LSIL and HSIL revealed loss of HPV diversity and domination by HPV-16 in HSIL samples. CONCLUSIONS: Deep sequencing resolves HPV genotype composition within multi-infected cervical cytology. Biodiversity analysis reveals loss of diversity and gain of dominance by carcinogenic genotypes in high-grade cytology. Metagenomic profiles may therefore serve as a biomarker of disease severity and a population surveillance tool for emerging genotypes.

E6 and E7 variants of human papillomavirus-16 and -52 in Japan, the Philippines, and Vietnam.

Human papillomavirus (HPV) has several intragenotypic variants with different geographical and ethnic distributions. This study aimed to elucidate the distribution patterns of E6 and E7 (E6/E7) intragenotypic variants of HPV type 16 (HPV-16), which is most common worldwide, and HPV-52, which is common in Asian countries such as Japan, the Philippines, and Vietnam. In previous studies, genomic DNA samples extracted from cervical swabs were collected from female sex workers in these three countries and found to be positive for HPV-16 or HPV-52. Samples were amplified further for their E6/E7 genes using type-specific primers and analyzed genetically. Seventy-nine HPV-16 E6/E7 genes were analyzed successfully and grouped into three lineages: European (Prototype), European (Asian), and African-2. The prevalences of HPV-16 European (Prototype)/European (Asian) lineages were 19.4%/80.6% (n = 31) in Japan, 75.0%/20.8% (n = 24) in the Philippines, and 0%/95.8% (n = 24) in Vietnam. The 109 HPV-52 E6/E7 genes analyzed successfully were grouped into four lineages, A-D; the prevalences of lineages A/B/C/D were, respectively, 5.1%/92.3%/0%/2.6% in Japan (n = 39), 34.4%/62.5%/0%/3.1% in the Philippines (n = 32), and 15.8%/73.7%/7.9%/2.6% in Vietnam (n = 38). The distribution patterns of HPV-16 and HPV-52 lineages in these countries differed significantly (P < 0.000001 and P = 0.0048, respectively). There was no significant relationship between abnormal cervical cytology and either HPV-16 E6/E7 lineages or specific amino acid mutations, such as E6 D25E, E6 L83V, and E7 N29S. Analysis of HPV-16 and HPV-52 E6/E7 genes can be a useful molecular-epidemiological tool to distinguish geographical diffusion routes of these HPV types in Asia.

[The study of gene variation and phylogenetic analysis of HPV16 E6 and E7 gene in Hubei, China].

To study the gene variation and the distribution of HPV16 variant in Hubei, China, DNA was extracted from cervical cancer tissue samples. The E6 and E7 genes of HPV16 were amplified and the PCR products were sequenced using E6- and E7-specific primers. Fortyseven cases were found mutations at nucleotide position 178 of HPV16 E6 gene in 80 cervical cancer samples. This mutation resulted in amino acid change from Asp to Glu. The rate of mutation at nucleotide position 178 of E6 gene was 58. 75%. Twenty two cases were found mutations at nucleotide position 647 of HPV16 E7 gene in 31 cervical cancer samples. This mutation resulted in amino acid change from Asn to Ser. The rate of mutation was 70.97%. These results showed that mutations at nucleotide position 178 of E6 gene, nucleotide position 647 of E7 gene of HPV16 in cerveical cancer samples were prevalent in Hubei, China. Phylogenetic analysis showed that Asian (As) variants of HPV16 are predominated in Hubei, China. European (Ep) varinats were also found in Samples in Hubei areas. None of Asian American (AA), African-1 (Af-1), African-2 (Af-2) variants of HPV16 was found in this region. Whether Asian (As) variants of HPV16 are more oncogenic and play a much more important role in the progress of cervical cancer than European (Ep) variants is not clear. More sequences of E6 and E7 gene in CIN and normal cervical tissue samples and study of the function of E6 and E7 protein of these HPV16 variants are needed to adress above question.