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

Jane Shen-Gunther,Luis Perez,Zhao Lai,Yufeng Wang,Graham M Poage,Tim H M Huang

doi:10.1186/s12864-017-3612-y

Abstract

BackgroundHuman 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.ResultsLow- 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.ConclusionsDeep 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.

Highlights

Human papillomavirus (HPV) is the carcinogen of almost all invasive cervical cancer and a major cause of oral and other anogenital malignancies
72 samples categorized as Low-grade squamous intraepithelial lesion (LSIL) (n = 43) and High-grade squamous intraepithelial lesion (HSIL) (n = 29) were analyzed and reported
Deep sequencing has provided a powerful lens through which to peer into viral communities and gain an understanding of a dynamic microcosm imperceptible with conventional methods

Summary

Introduction

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. Its cause remained a mystery for two millennia until 1983 when zur Hausen and colleagues isolated and cloned HPV-16 from cervical carcinoma [2]. The HPV genome is a ~8,000 base pair (bp), doublestranded, circular DNA. HPV classification is based on the L1 gene where a difference of >10% in the viral sequence defines a different genotype [6]. With the advent of sequencing technologies, the list of papillomavirus (PV) genotypes has grown to 333 with 202 types isolated from humans and 131 from animals

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