Summary auto-generated
This study analyzed HPV-16 (human papillomavirus type 16) variants from five penile carcinoma biopsies obtained from Ugandan patients, a region with high rates of genital cancer. Researchers sequenced the E6/E7, L1, and long control region (LCR) of the viral genome and performed phylogenetic analysis. All five isolates belonged to the African-1 (Af1) lineage based on LCR sequences. However, four isolates represented a new subclass designated Af1-u, characterized by distinctive mutations in the E6 gene at positions 10 and 14. The E6 gene showed the greatest sequence variation among tested regions, while E7 and L1 were highly conserved. A novel mutation in one sample caused loss of a zinc-binding motif in the E6 protein. Critically, PCR and Southern blot analysis revealed that four of five samples contained integrated HPV-16 DNA with disruption of the E2 gene—integration into human chromosomal DNA rather than episomal (free circular) viral DNA. This is the first report documenting HPV-16 integration with E2 disruption in male genital cancers, paralleling patterns seen in cervical cancer where E2 disruption increases expression of oncogenic E6 and E7 proteins.
Key findings
- Four of five Ugandan penile carcinoma samples contained HPV-16 integrated into human DNA with disruption of the E2 gene, the first such report in male genital cancers
- A new HPV-16 subclass (Af1-u) was identified with characteristic mutations at amino acid positions 10 and 14 in the E6 protein
- The E6 gene showed the greatest sequence variability among tested genomic regions, while E7 and L1 were highly conserved across isolates
- One sample contained a novel E6 mutation causing loss of a zinc-binding motif, with unknown implications for oncogenic activity
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Abstract
Sequence variations in the E6/E7 (nt 34-880) and the L1-(nt 6584-7035) ORFs, and in the long control region (LCR) (nt 7289-93) of human papillomavirus type 16 (HPV-16) were analysed in five penile carcinoma biopsies obtained from Ugandan patients. Uganda is a country with a high incidence of genital cancers. All five isolates were classified as members of African-1 lineage (Af1) by phylogenetic analysis based on LCR sequences. The E6 gene phylogenetic analysis, however, showed that four isolates fell into a new subclass designated Af1-u. This subclass, characterized by three point mutations located at the 5' end of the E6 gene with resulting changes in amino acids at positions 10 and 14, is distinguishable from the Af1 class by the absence of synonymous mutations at nt 286 and 289. The nonsynonymous substitution at nt 335 was present in three out of five samples. The E6 Af1 mutation pattern was present in only a single Ugandan HPV-16 isolate. Nucleotide sequence analysis of the E7 and L1 regions did not allow any Af1 subclass identification. The physical state of the viral DNA in these samples was characterized by PCR and Southern blot analysis. Oligonucleotides which enable amplification of the full length E2 region (nt 2734-3872) failed to amplify the target sequence in four out of five samples, suggesting disruption of the E2 ORF and integration of the HPV genome into the human DNA. Southern blot analysis confirmed the virus integration status. Our results contribute to the characterization of the HPV-16 'African lineages' with the identification of the Af1-u subclass; furthermore, this is also the first report showing that in male genital cancers HPV-16 is integrated into the human genome with disruption of the E2 ORF.