Fifteen new nucleotide substitutions in variants of human papillomavirus 18 in Korea : Korean HPV18 variants and clinical manifestation

Abstract

High-risk human papillomavirus (HPV) infection is an essential factor for the development of cervical cancer. HPV18 is the second most common carcinogenic HPV type following HPV16, but the lineages of HPV18 have been less well studied than those of HPV 16. The purpose of this study was to analyze the nucleotide variants in the E6, E7, and L1 genes of HPV18, to assess the prevalence of HPV18 variants in Korea and to explore the relationship between HPV18 genetic variants and the risk for cervical cancer.A total of 170 DNA samples from HPV18-positive cervical specimens were collected from women admitted to a secondary referral hospital located in Seoul. Among them, the lineages of the 97 samples could be successfully determined by historical nomenclature.All the studied HPV 18 variants were lineage A. Sublineages A1 and A4 comprised 91.7% (89/97) and 1.0% (1/97), respectively. Sublineages other than A1 or A4 comprised 7.2% (7/97). We identified 15 new nucleotide substitutions among 44 nucleotide substitutions: C158T, T317G, T443G, A560G, A5467G, A5560C, A5678C, A6155G, G6462A, T6650G, G6701A, T6809C, A6823G, T6941C and T6953C. Among them, 6 substitutions at positions 317, 443, 5467, 5560, 6462, and 6823 resulted in amino acid changes (E6: F71L and N113K; L1: H13R, H44P, A345T, and N465S, respectively). The pathologic results were classified as normal in 25.8% (25/97) of the women, atypical squamous cells of undermined significance (ASCUS) in 7.2% (7/97), cervical intraepithelial neoplasia (CIN) 1 in 36.1% (35/97), CIN2/3 in 19.6% (18/97), and carcinoma in 12.4% (12/97). There was no significant association between the HPV18 sublineages and the severity of pathologic lesion or the disease progression.This study is the first to analyze the distribution of HPV18 variants in Korean and to associate the results with pathologic findings. Although the HPV18 variants had no significant effect on the degree and progression of the disease, the newly discovered nonsynonymous mutation in L1 might serve as a database to determine vaccine efficacy in Korean women.

Keywords: Cervical cancer; E6, E7 and L1 genes; Human papillomavirus (HPV) 18; Lineage; Variants.

Fig. 1

Nucleotide sequence variations in the HPV E6-E7-L1 isolates in Korean women. ^*Previously reported: N, No ^†Grade of cervical lesions: ① Normal, ② ASCUS, ③ LSIL/CIN1, ④ HSIL/CIN2 and 3, ⑤ Cancer ^‡L1 secondary structure: H, helix; C, coil; S, strand Abbreviations: AA, Asian-Amerindian; E, European; ASCUS, Atypical squamous cells of undetermined significance; LSIL, Low-grade squamous intraepithelial lesion; HSIL, High-grade squamous intraepithelial lesion; CIN, Cervical intraepithelial neoplasia; na, not assessed; aa, amino acid. E6, E7 and L1 nucleotide positions at which variations were observed are written vertically across the top. Amino acid translations and immunodominant loop structures of L1 are shown at the bottom. The GenBank accession numbers of the newly discovered sequences are on the left. The phylogenetic groupings based on the analysis of E6-E7 are indicated. For each variant sequence, the positions that do not vary relative to the HPV reference are marked with a blank. The regions where sequencing failed are marked with an X. The cervical lesion grade for each variant is shown on the right

Fig. 2

Phylogenetic tree of the HPV18 variants by the Maximum Likelihood method. The evolutionary history was inferred using the Maximum Likelihood method with 1000 bootstraps in a Tamura-Nei model. All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). Numbers near the line indicate bootstrap values. Evolutionary analyses were conducted in MEGA X