Analysis of Sequence Variation and Risk Association of Human Papillomavirus 52 Variants Circulating in Korea

Introduction Human papillomavirus (HPV) 52 is a carcinogenic, high-risk genotype frequently detected in cervical cancer cases from East Asia, including Korea. Materials and Methods Sequences of HPV52 detected in 91 cervical samples collected from women attending Seoul St. Mary’s Hospital were analyzed. HPV52 genomic sequences were obtained by polymerase chain reaction (PCR)-based sequencing and analyzed using Seq-Scape software, and phylogenetic trees were constructed using MEGA6 software. Results Of the 91 cervical samples, 40 were normal, 22 were low-grade lesions, 21 were high-grade lesions and 7 were squamous cell carcinomas. Four HPV52 variant lineages (A, B, C and D) were identified. Lineage B was the most frequently detected lineage, followed by lineage C. By analyzing the two most frequently detected lineages (B and C), we found that distinct variations existed in each lineage. We also found that a lineage B-specific mutation K93R (A379G) was associated with an increased risk of cervical neoplasia. Conclusions To our knowledge, we are the first to reveal the predominance of the HPV52 lineages, B and C, in Korea. We also found these lineages harbored distinct genetic alterations that may affect oncogenicity. Our findings increase our understanding on the heterogeneity of HPV52 variants, and may be useful for the development of new diagnostic assays and therapeutic vaccines.


Introduction
Human papillomavirus (HPV) is the major causative agent of cervical cancer, a leading cause of death among women worldwide [1]. The virus genome is divided into three functional regions: an upstream regulatory region, an early region, and a late region. The upstream regulatory region is a non-coding region, referred as the long control region (LCR), which regulates transcriptional and replication activities. In comparison, the early and late regions are coding regions. Early regions (E1, E2, E4, E5, E6 and E7) encode for non-structural proteins while late regions (L1 and L2) encode for structural proteins [2]. HPV is markedly heterogeneous with more than 200 genotypes which are classified into types, lineages, and sub-lineages based on the L1 sequence. The L1 sequences among different types differ by at least 10%, and those of lineages differ by >1% [3,4]. The persistence of HPV contributes to the progression of cervical infection to cervical cancer. In particular, oncogenicity varies according to the HPV genotype, as well as the lineage of some genotypes [4][5][6]. Eight most common high-risk HPV genotypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52 and HPV58) are responsible for 91% of cervical cancers [7,8]. Of these, HPV52 is a high-risk genotype and one of the nine HPV types (HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58) targeted by the recent Food and Drug Administration (FDA) approved HPV 9-valent vaccine [9].
Given that HPV52 is recognized as a high-risk genotype commonly found in cervical cancers from East Asia [10][11][12], we attempted to characterize HPV52 variants circulating in Korea and to investigate their association with cervical cancer development.

Cervical samples
Altogether, 91 cervical cytology/tissue samples that had tested positive for HPV52 were used for this study. These samples had been collected as part of the routine clinical management at Seoul St. Mary's Hospital (Seoul, Korea) and all were treatment-naive. This study was approved by the institutional review board of the Catholic University of Korea, College of Medicine and the participants provided written informed consent. Pathologic features of patients are summarized in Table 1. The quality of DNA extracted from cytology/tissue samples was assessed by amplifying a 932-bp fragment of the long-control region (LCR). The HPV genotype was ascertained by demonstrating a nucleotide sequence similarity of >90%, compared with the HPV52 prototype (GenBank accession no. X74481).
Nucleotide sequencing E6, E7, L1 and LCR sequences were amplified with long-or short-fragment polymerase chain reaction (PCR). Long-fragment PCR was performed on good-quality samples with primers 5 0 -ATG TCC ATT GAG TCA GGT CC-3 0 and 5 0 -TGC ATT TTC ATC CTC GTC C-3 0 . When the first-round PCR product was not strong enough for sequencing, a second-round PCR was performed using inner primers 5 0 -GGT CCT GAC ATT CCA TTA CC-3 0 and 5 0 -CCT CTA CTT CAA ACC AGC CT-3 0 when necessary 0 (S1 Table). Each PCR was conducted in a 50-μL reaction mixture containing 1 unit of Phusion Hot Start II High-Fidelity DNA Polymerase  Table). Each PCR was conducted in a 30-μL reaction mixture containing 0.75 unit of HotStarTaq Plus DNA Polymerase (QIAGEN, Hilden, Germany), 200 μM of dNTPs and 0.25 μM of each primer. An aliquot of 3 μL of extracted DNA was added as template. The thermal cycling began with a 5-min initial denaturation and enzyme activation at 95˚C, followed by 35 cycles of 1-min denaturation at 94˚C, 1-min annealing at 58˚C and 40-sec extension at 72˚C, and ended with an 8-min final extension at 72˚C. PCR products were sequenced from both directions and analyzed using Seq-Scape software (version 2.5, Applied Biosystems, Foster City, CA, USA). Repeated sequencing was performed as a confirmation when mutations occurred only once.

Statistical analysis
Statistical analysis was performed using a commercially available statistical software package (SPSS statistical software version 18.0 [SPSS Inc, Chicago, IL, USA]). Fisher's exact test and logistic regression analysis were used to analyze categorical data. The level of significance was set at P < 0.05.

Lineage identification
Altogether, four HPV52 variant lineages were identified based on the phylogenetic tree topology (Fig 1).

Discussion
HPVs are circular double-stranded DNA viruses that consist of heterogeneous variants with different pathogenicities [4,14]. HPV52 is one of the most frequently detected carcinogenic high-risk genotypes in East Asia [15][16][17] and is one of the genotypes targeted by the recent, USA FDA-approved HPV 9-valent vaccine [9]. However, only a few studies on the pathogenicity of HPV52 are available [18]. In this regard, we attempted to achieve three aims. Firstly, we aimed at identifying HPV52 variants and lineages based on E6, E7, L1 and LCR sequences. Secondly, we attempted to delineate lineage-specific variations. Lastly, we sought to examine the risk association of various lineages, variants and mutations. As a result, we identified four HPV52 variant lineages (A, B, C and D), with lineage B (86.8%) being the most frequently detected, followed by lineages C (6.6%), A (5.5%), and D (1.1%). By further analyzing the two most frequently detected lineages, we found distinct sequence variations in each lineage. Of note, one of lineage B-specific mutations was found to associate with a higher risk for cervical cancer.
Our findings of the two most frequently detected lineages in HPV52, B and C, also confirm previous observations [19,20]. These two lineages are associated with high-grade lesions [19,21], and we have shown that they harbor a number of lineage-specific variations (Tables 2  and 3). Lineage B-specific mutations included the most frequently detected nonsynonymous mutation K93R (A379G) in E6, while lineage C-specific mutations included an E6 nonsynonymous mutation (L83V, concurrent mutations of C348G and G350T) and five E7 nonsynonymous mutations, S52D (concurrent mutations of A706G and G707A), Y55D (T727G), H61Y (C733T), D64N (G742A) and L99R (T848G)). Our findings suggest that lineage-specific mutations may contribute to the carcinogenicity of each HPV52 lineage. E6 is an oncogene that interacts with a well-known tumor suppressor, TP53, increasing the risk for the accumulation of genetic changes [22] and inhibiting cellular responses such as cell cycle arrest, induction of apoptosis and DNA damage repair [23,24]. K93R (A379G) is a nonsynonymous mutation located in the E6 oncogene, which may have a specific role in carcinogenesis: it is not only the most frequently detected variation [25], but is also independently associated with high-grade lesions [26]. An alteration at nucleotide position 350 in E6 was found for both lineages B and C. However, lineage C harbored an additional alteration at nucleotide position 348, yielding a nonsynonymous mutation, L83V. L83V have been reported not only in HPV52, but also in HPV16 and HPV33 [25,27,28]. In addition, an association of HPV16 L83V with high-grade lesions has been shown [29], suggesting that it may contribute to the pathogenicity of lineage C [19]. It is noticeable that E7 nonsynonymous mutations (S52D, 55D, H61Y, D64N and L99R) were harbored by lineage C, but not by lineage B. E7 interacts with the retinoblastoma (Rb) protein, causing uncontrolled cell division and inactivating its function as a tumor suppressor [30,31].

Conclusions
HPV52 is the second to fifth most frequently detected high-risk HPV genotype in Korea [15,[32][33][34]. Our data demonstrated for the first time that HPV52 lineages (B and C) circulating in Korea harbor distinct genetic alterations that may affect pathogenicity. We also observed that most of the cervical samples (ranging from normal cervix to SCC) were infected with HPV52 lineages B or C that carried putative high-risk mutations. Our findings may provide a useful basis to understand the heterogeneity of HPV52 variants in Korea, and to assist the development of diagnostic assays and vaccines.
Supporting Information S1