65ª Reunião Anual da SBPC

C. Ciências Biológicas - 8. Genética - 2. Genética de Microorganismos

GENETIC VARIABILITY IN CAPSID L1 GENE OF RARE HUMAN PAPILLOMAVIRUSES (HPV) FOUND IN CERVICAL LESIONS OF WOMEN FROM NORTHEASTERN BRAZIL

Ana Pavla Almeida Diniz Gurgel - Department of Genetics- Federal University of Pernambuco Bárbara Simas Chagas - Department of Genetics- Federal University of Pernambuco Carolina Medeiros do Amaral - Department of Genetics- Federal University of Pernambuco Jacinto da Costa Silva Neto - Department of Histology and Embryology - Federal University of Pernambuco Maria Thereza Cartaxo - Pediatric Oncohematology Center – University of Pernambuco Antonio Carlos de Freitas - Department of Genetics- Federal University of Pernambuco(Orientador)

INTRODUÇÃO:

Cervical cancer is the second major cause of female cancer worldwide, with more than 529,000 new cases diagnosed and 275,000 deaths. Particularly in Brazil, cervical cancer is the third most common female cancer. There have been several studies that report prevalence and genetic variability of the HPV16, HPV18, HPV31 and HPV58 genotypes. However, few studies showed the extent of the variability of rare HPV types in infected women. Rare HPV types involved in cervical cancer may be explained by the genetic variability, mainly when these changes are inserted in linear B-cell or T-cell epitopes, which theoretically allow the escape recognition by the innate immune system. Furthermore, it is still unknown the role they play in single infections or co-infections that result in cervical lesions and cervical cancer.

OBJETIVO DO TRABALHO:

The aim of this study was to investigate the genetic variability of the capsid L1 gene of the HPV53, 54, 56, 61, 62, 70 and 81 viruses that were found in cervical lesions of women from Northeastern Brazil.

MÉTODOS:

A total number of 263 women agreed to participate in the study. DNA was extracted by using the Dneasy Blood and Tissue Kit 135 (Qiagen). HPV DNA detection was performed by using polymerase chain reaction (PCR) with degenerate MY09/11 primers. The reactions were performed in a final volume of 25µL containing 150ng of DNA, 1.5 Mm of MgCl2, 50 µM of each dNTPs, 20 pmol of each primer, 1 unit of Taq DNA Polymerase and 1X buffer. The PCR cycling conditions were as following: initial denaturation at 95ºC for 5 minutes, 35 cycles of denaturation at 95 ºC for 30 seconds, annealing at 55ºC for 30 seconds, elongation at 72ºC for 40 seconds and a final extension at 72ºC for 10 minutes. The positive HPV DNA samples were sequenced by using ABI PRISM BigDyeTM Terminator Cycle Sequencing v 3.1 (Applied Biosystems®). The studied rare HPVs were then evaluated in order to verify nucleotide divergence relative to the L1 nucleotide sequences of the HPV53, 54, 56, 61, 62, 70 and 81 prototypes. Sequence comparisons were performed by using Basic Local Alignment Search Tool (BLAST) and multiple alignments were carried out by CLUSTALW (Mby ega 5, Beta version) program. The putative impact of the variability in L1 gene of rare HPV types was estimated in silico by predicting the B-cell and T-cell epitopes using BcePred, ProPred and ProPred I servers.

RESULTADOS E DISCUSSÃO:

Genetic variability in L1 gene of the rare HPV types showed thirty nucleotide changes, eight of which were detected for the first time in this study. The analysis of the variability of the L1 gene of HPV53 revealed the C6942T mutation. This mutation leads to amino acid changes involving P430S, which are embedded in B-cell and T-cell epitope. Concerning to HPV70, the analysis showed the mutation point A6886G that leads to the T433A amino acid change, which is embedded in the T-cell and B-cell epitope binding regions. The L1 genetic variability of HPV54 showed A6622T and T6625C mutation points that lead to Q313H and S333T amino acid changes in the L1 protein sequence, respectively. An analysis of the genetic variability of L1 of HPV81 showed a new synonymous mutation at the A7148G position. In addition, two synonymous mutations in HPV56 A6666G and A6987G were observed. Genetic variability in L1 protein of HPV62 showed nine nucleotide exchanges, in which five of them are non-synonymous mutations. Among these, two new mutations were found at 6838C and A6921G, which led to E354D and Q382R amino acid changes, respectively. The results of this research suggest that rare HPV types are involved in cervical lesions and some of these variants are inserted in B-cell and T-cell epitopes.

CONCLUSÕES:

Genetic variability in L1 genes of rare HPV types showed several synonymous and non-synonymous mutations, which can lead to altered immune responses. Further investigation of prevalence and variability with regard to rare HPV types can clarify the role of this virus in infection and carcinogenesis.

Palavras-chave: Rare Human Papillomaviruses (HPV), HPV variants, L1 gene.