65ª Reunião Anual da SBPC

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

HAPLOTYPE ASSOCIATION BETWEEN POLYMORPHISMS IN THE MAJOR CAPSID L1 GENE OF HUMAN PAPILLOMAVIRUS (HPV) TYPE 16 AND RISK TO DEVELOP CERVICAL LESIONS

Kamylla Conceição Gomes Nascimento - Department of Genetics- Federal University of Pernambuco Ana Pavla Almeida Diniz Gurgel - Department of Genetics- Federal University of Pernambuco Marcus Vinícius Cardoso - Department of Genetics- Federal University of Pernambuco Jacinto da Costa Silva Neto - Department of Histology and Embriology - Federal University of Pernambuco Maria Thereza Cartaxo - Pediatric Oncohematology Center – University of Pernambuco Antonio Carlos de Freitas - Prof. Dr. Orientador - Department of Genetics- Federal University of Pernambuco

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. It is well-established that genetic and environmental factors are involved in pre-cancerous lesions and cervical cancer. Persistent infections caused by Human Papillomavirus (HPV) are recognized as a main cause of pre-cancerous lesions and cervical cancer. However, although a large number of sexually active women are infected with HPV, a few of them develop pre-cancerous lesions or cervical cancer. Therefore, other genetics and environmental factors must be involved in the development of cervical lesions and cervical cancer, such as individual lifestyle, polymorphisms in host genes related to apoptosis and immune system, and intratypic variability of HPV. Several studies have been demonstrated that genetic variants of HPV16 are associated with persistence and progression of pre-cancerous lesions to cervical cancer. However, there were no studies concerning polymorphic regions of L1 gene from HPV16 as molecular markers of prediction to cervical lesions or cervical cancer in women infected with HPV16 and without cervical lesions.

OBJETIVO DO TRABALHO:

The present study aims to report haplotype association between L1 gene polymorphisms and high risk in developing cervical lesion in women from Northeastern Brazil.

MÉTODOS:

A total number of 51 women agreed to participate in the study. DNA was extracted by using the Dneasy Blood and Tissue Kit 135 (Qiagen) in accordance with the manufacturer’s instructions. HPV DNA detection was performed by using polymerase chain reaction (PCR) with degenerate primers MY09/11. The reactions were performed in a final volume of 25µL containing 150ng of DNA, 1.5 Mm of MgCl2, 50 µM of each DNTp, 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 PCR products were run on the agarose gel (1%). The positive HPV16 samples were purified with the Invisorb® Fragment Cleanup (Invitek) kit and sequenced (in duplicate) by using ABI PRISM BigDyeTM Terminator Cycle Sequencing v 3.1 Ready Reaction (Applied Biosystems®). The viral polymorphisms included in this study are located within L1 gene at position 6693-6864. The genetic frequency and association analysis between the comparison groups were performed with UNPHASED v.3.121. All tests were two-tailed and the level of significance for all statistical results was set at p < 0.05.

RESULTADOS E DISCUSSÃO:

A6693C and A6801T viral polymorphisms distribution were in Hardy-Weinberg equilibrium in case and control groups (p>0.05). There were no significant statistical differences in genotype distribution or allele frequencies of the polymorphisms between case and control subjects (p>0.05). In addition, haplotype test were carried out for genotyping data of A6693C and A6801T polymorphisms found within L1 gene of HPV16. The results showed an association between A6693C and A6801T (CA alelles, p=0.02, Odds ratio 4.57). In the present study, it was found association between L1 gene polymorphism and high risk to develop cervical lesion in women infected with HPV16. The odds ratio of these polymorphisms was 4.57, indicating the high risk to develop cervical lesions in women infected with the HPV16 variants. To the best of our knowledge, this is the first study that demonstrated association between haplotypes in L1 gene and risk to develop cervical lesions. A6693C and A6801T are polymorphisms located in L1 gene of HPV16. Intratypic variants in HPV16 L1 gene can be related to the escape recognition by the innate immune system.

CONCLUSÕES:

Thus, this study reports the haplotype association between A6693C and A6801T viral polymorphisms and high risk to develop pre-cancerous lesions in women infected with HPV16 variants.

Palavras-chave: Human Papillomavirus Type 16, pre-cancerous lesions, viral polymorphisms.