Human papillomavirus partial L2 gene variants in the Barrett’s metaplasia-dysplasia-adenocarcinoma sequence

TL;DR

This study examines HPV gene variants in the progression of Barrett’s esophagus to cancer, finding that HPV-16 is most common.

Contribution

The study identifies the prevalence of HPV-16 over HPV-18 in Barrett’s metaplasia-dysplasia-adenocarcinoma sequence.

Findings

32 out of 33 HPV partial L2 genes sequenced were HPV-16.

Only one specimen was identified as HPV-18.

Phylogenetic analysis highlights HPV-16's dominance in the disease progression.

Abstract

High-risk human papillomavirus (HPV) genotypes 16 and 18 are associated with Barrett’s dysplasia and esophageal adenocarcinoma. We sequenced 33 HPV partial L2 genes from Australian esophageal specimens. Phylogenetic analysis showed 32 were HPV-16 and one was HPV-18, underscoring the predominance and importance of HPV-16.

Figures

Phylogenetic tree based on the partial L2 gene sequences of HPV. Raw sequence reads from forward and reverse primers were assembled and trimmed to generate consensus sequences for each sample using BioEdit software. Consensus sequences were compared with published sequences in the NCBI database using BLASTn. Multiple sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) with available sequences from GenBank. The alignment included 47 nucleotide sequences, with codon positions 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated, resulting in a final data set of 81 positions. The phylogenetic tree was constructed using the Maximum Likelihood method in MEGA X (7) with 1,000 bootstrap replications, applying the maximum composite likelihood model. Evolutionary analyses were conducted in MEGA X. All HPV16 variants were highly similar, with pairwise sequence similarity ranging from 99.79% to 100%, mainly because of the sequence length.

Tables

TABLE 1. PCR conditions for amplification of HPV L2 gene regions

Component	HPV-16 (HQ644299)	HPV-18 (GQ180792)	Thermal cycling conditions
Forward primer (5’→3’)	AAC CGA AAT CGG TTG AAC CG	GGA GTA ACC GAA AAC GGT	Initial denaturation: 95°C, 2 min35 cycles: - Denaturation: 95°C, 30 s - Annealing: 57°C, 30 s - Extension: 72°C, 1 min Final extension: 72°C
Reverse primer (5’→3’)	TGA TGT GTA TGT AGA CAC AGA C	CAT ACA TGC ATA CAC AAA AGC
Target region	Partial L2 gene	Partial L2 gene
Reference sequence	HQ644299	GQ180792

Full text

ANNOUNCEMENT

Oncogenic viruses are responsible for a substantial percentage of global cancer cases (1). Human papillomavirus (HPV), a key player, is linked to approximately 30% of virus-related cancers (2). HPV is a small, double-stranded DNA virus that belongs to the Papillomaviridae family with an ~8 kb genome divided into early (E), late (L), and long control (LCR) regions; types are classified based on <90% L1 gene nucleotide similarity, subtypes on 90–98%, and variants on >98% similarity (3). The L2 gene was selected in this study because its partial conservation made it a suitable target for broad primers, while its higher divergence provided better variant discrimination than L1 (4).

In esophageal adenocarcinoma (OAC), studies have indicated that high-risk HPV is associated with 25% of malignancies (5, 6). In this study, we report the partial L2 genes of HPV strains detected from OAC cases from Bankstown-Lidcombe Hospital in Sydney, Australia, and compare them phylogenetically to previously reported strains. Viral genome sequences are essential for understanding the genetic diversity, evolution, and epidemiology of HPV.

From 2012 to 2014, we collected 33 biopsies from patients with Barrett’s metaplasia–dysplasia–adenocarcinoma (median age 46, range 42–79). Samples were homogenized, and viral DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, USA). DNA quality and concentration were assessed with a NanoDrop spectrophotometer. The partial L2 region was amplified by PCR with type-specific primers (Table 1), visualized on agarose gels, and purified (Qiagen, USA). Amplicons (90–100 bp) were sequenced bidirectionally by the Sanger method at the Ramaciotti Centre for Genomics (UNSW, Sydney). Consensus sequences were compared with GenBank references, showing >99% similarity, and the most closely related strains were used for phylogenetic analysis. A maximum likelihood tree was constructed in MEGA X to determine evolutionary relationships.

Sequences showed >99% similarity with GenBank references. One sequence clustered with HPV-18 strains CU9 (Thailand) and Qv03132 (USA), while 32 clustered with HPV-16 strains CU2/CU4 (Thailand) and African-1 (USA) (Fig. 1).

Phylogenetic tree based on the partial L2 gene sequences of HPV. Raw sequence reads from forward and reverse primers were assembled and trimmed to generate consensus sequences for each sample using BioEdit software. Consensus sequences were compared with published sequences in the NCBI database using BLASTn. Multiple sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) with available sequences from GenBank. The alignment included 47 nucleotide sequences, with codon positions 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated, resulting in a final data set of 81 positions. The phylogenetic tree was constructed using the Maximum Likelihood method in MEGA X (7) with 1,000 bootstrap replications, applying the maximum composite likelihood model. Evolutionary analyses were conducted in MEGA X. All HPV16 variants were highly similar, with pairwise sequence similarity ranging from 99.79% to 100%, mainly because of the sequence length.

The predominance of HPV-16 among the samples aligns with global epidemiological trends, highlighting its significant role in oncogenesis (8). The clustering patterns observed could inform future public health strategies and vaccination programs. Additionally, the identification of HPV-18, although less frequent, emphasizes the need for comprehensive screening and monitoring to address all high-risk HPV types effectively.