HPV6 and HPV11 Genome Methylation in Condyloma AccuminatumMeasured by Bisulfite Sequencing

Abstract

To the Editor: Condyloma acuminatum (CA, or genital warts) is a highly contagious and common sexually transmitted disease and caused by the infection of some types of human papillomavirus (HPV), especially types 6 and 11.1 Methylation is the most popular modification in human genome and has been observed to be correlated with human diseases.2 Moreover, it was known that methylation of partial region in multiple HPV genomes, including 16, 18, 30, and 45, was correlated with cervical cancer,3,4 another disease in women caused by HPV infection. All these facts made us hypothesize that methylation of HPV genome might also be correlated with CA. However, the HPV methylation status in CA is unknown. In this pilot study, we investigated the HPV genome methylation status in patients with CA by bisulfite sequencing. To this end, multiple genital skin tissues from typical male patients with CA with giant genital warts were collected from surgery and genotyped for HPV infection by Polymerase Chain Reaction (PCR) and xMAP (Luminex, Austin, TX; primer and probe set available on request). All patients were diagnosed based on typical clinical presentations5 and further confirmed by biopsy findings. From these samples, 4 patients with HPV6 infection and 3 with HPV11 were chosen for this study. DNA was isolated by standard phenol–chloroform method and treated with freshly prepared bisulfite using the EZ DNA methylation kit (Zymo Research, Orange, CA) according to the manufacturer's protocol. In bisulfite treatment, unmethylated C was converted into U, which was then converted to T in following PCR, whereas methylated C remained unmodified. Because HPV genome methylation in cervical tissue showed a gene-specific manner,3,4 which meant that the methylation in one segment could represent the situation in whole gene, we designed one PCR assay (see Table S1, Supplemental Digital Content 1, https://links.lww.com/AJDP/A65) for each protein-coding gene and upstream regulatory region by Methprimer6 (http://www.urogene.org/methprimer/index1.html). All segments were amplified by Taq DNA polymerase (Takara, Dalian, China) and directly sequenced in AB 3730 sequencer (Thermo Fisher, Waltham, MA) after purification, which was an accurate approach, and had been widely used in previous studies.7 This study was approved by the Institutional Ethics Committee, and informed consent was obtained from all individuals. In this study, 76 and 54 CpG sites were surveyed for HPV6 and 11 genomes, respectively, which account for ∼35–48% of total CpG sites, and the chromatograms for each CpG sites were inspected visually. None of the CpG sites displayed a clear C peak, which indicated that no methylation was observed in HPV6 and 11 genomes from CA tissues, and was consistent with recent report in HPV11 and 6 from respiratory8 and laryngeal9 papillomas tissues, respectively. Although the normal skin tissue was not available for control (persons with HPV infection but without CA symptom) in this study, we still could deduce that the methylation in normal genital tissue was also absent because of the hypomethylation of HPV genome in cervix without neoplasia3,4 and the lack of genes coding for DNA methyltransferase in HPV genome. Another concern was the relatively small sample size and sample bias (only male patients included) in this study. Because most patients were prone to receive pharmacotherapy instead of surgery, it was difficult to obtain more tissues. Further investigation with larger sample size and female patients might generalize our conclusion. In another side, because DNA methylation level was relatively similar among the different patients with same disease, it could be speculated that other patients with CA might also present hypomethylation in HPV genome. All these facts indicated that the methylation of HPV genome was not changed during the development of CA, which might indicate that the methyltransferase system still worked properly in CA tissues.