L1 Gene Molecular Variation of Human Papilloma Virus Type 16 from Cervical Cancer Patient

Background: Cervical cancer is the second most cancer in the world after breast cancer, this cancer is caused by infection of high risk Human Papillomavirus (HPV) type 16. It is often found in cervical cancer whose genome structure is composed of L1 proteins. L1 protein makes up the viral capsid that has an important role to infect the cervical epithelium. Several studies have found the differences in HPV nucleotides variants that lead to changes in amino acids that can disrupt the structure, the nature function of the virus itself, and ultimately lead to changes in biological functions including host immunological recognition. Variation of the L1 gene will also affect the effectiveness of existing vaccines. Methods: This research is a descriptive study conducted at the laboratory of microbiology at the Faculty of Medicine at Andalas Padang University from February to August 2018 which aims to look at the molecular variations of the L1 HPV type 16 gene and see phylogenic kinship. Results: This study obtained SNPs (Single Nucleotide Polymorphism) on all HPV 16 samples in the form of C / G (6240), A / G (6432), T / G (6686), C / T (6823) and insertion of nucleotide bases ACT (6901) ) and followed by GAT base deletions (6953) variations occurring along the observed sample isolate sequences. Conclusion: There are molecular variations of the L1 HPV type 16 gene which can cause different host immune responses. Phylogenic kinship of HPV type 16 isolate in Riau is close to Asian-American isolate.


Introduction
Cervical cancer is the second most malignancies in women in the world (Bruni et al., 2017). Concerning the epidemiology, it is estimated there are 528,000 new cases and 266,000 deaths in 2012 (Farley et al., 2015). If the number of cases continues to increase, it is estimated that by 2025 cervical cancer in the world will reach 20 million new cases. The main risk factor for cervical cancer is related to Human papillomavirus (HPV) infection in the cervical epithelium (Akyar et al., 2013;Manga et al., 2015;Nurcahyanti, 2016).
It is estimated that 5% of cancers in humans are caused by Human papillomavirus (HPV) infections, some of these cancers originate from cervical cancer. Type 16 HPV is a major cause of cervical cancer, with percentage, respectively, 45.5% in worldwide and 60% in Indonesia (Bruni et al., 2017). It is reported that 70% of cervical cancer caused by the infection of oncogenic type of HPV (high risk), and it is associated with anogenital cancers in men and women, such as cancer of the penis, vulva, vagina, anal, and oropharyngeal cancer (Setiawati, 2014). Approximately there are 100 different subtypes of HPV with different variations in potency and oncogenic, and which speci cally infecting anogenital area are HPV types 16, 18, 31, 33, 35, 39, 45, 52, 52, 56, 58, 66, and 69 ( Faridi et al., 2011) In the study of cervical cancer samples, it is proved the presence of HPV deoxyribonucleid acid (HPV DNA) was found in 99.7% cases and was dominated by types 16, 18, 31 and 45, this shows the role of high-risk HPV in the development of cervical cancer (Lipinwati, 2014).
Human papillomavirus (HPV) is a double-stranded, non-envelope DNA viruses measuring around 7,200-8,000 base pairs. It enveloped in capsid protein which is composed of L1 protein (major capsules) and L2 protein (minor capsid). The viral genome is divided into 3 regions, namely non-coding regions (initial regulation) or also known as long-control regions (LCR) of 400-1.000 bp, the second region is an initial region consisting of an Early Protein Open Reading Frame (ORF) such as E1, E2, E4, E5, E6 and E7, the third region is the region of late protein that encodes L1 and L2 proteins which form a viral capsids or viral envelopes, in which L1 and L2 capsids play a role in the transmission of Identi ed HPV-16 DNA isolates were ampli ed using three primer pairs, Table 1 self-designed with PCR settings, Initial denaturation at 94 C for 5 minutes, Denaturized at 95 C for 30 seconds, Annealing at 56 C for 45 seconds, Extension at 72 C for 1 minute Post Extension at temperature of 72 C for 10 minutes, repeatedly for 35 cycles. To see the results of the Polymerase Chain Reaction (PCR), an electrophoresis device using 0.8% agarose gel was used. The PCR results are processed through a sequencing process to obtain nucleotide base sequence sequences from the HPV-16 L1 gene. The process of analyzing the results of sequencing data use Bioedit software (version 7.0.4.1), NCBI BLAST, CLUSTALX, and for the construction of phylogenetic trees we use software and MEGA 6 so that phylogenetic trees can be arranged. Phylogenetic trees were made based on sequences of HPV-16 and their genetic relationship by comparing the sequence of nucleotide bases from the HPV-16 L1 gene isolates of cervical cancer patients from another countries.

Results
From the results of the ampli cation using three pairs of forward and riverse primers, DNA fragments of different sizes were obtained, primer I 718 bp, primary II 1000 bp, primary III 751 bp (Fig. 1a, 1b, 1c). The sequence data of nucleotide base pairs isolates samples obtained from the sequencing results were compared with the reference sequences of the HPV-16 L1 gene in the GenBank (K02718.1). After being analyzed, we obtained a sequence size of 1596 bp. Based on sequencing analysis, it was found that all isolates sample experienced SNPs in their DNA sequences, where not all nucleotide bases match with the reference sequence HPV-16 (K02718.1. These SNPs have the potential to affect the amino acid composition ( Table 2). These results are processed in the CLUSTALX and MEGA 6 programs. We analyze genetic relationship by making phylogenic trees of HPV-16 L1 gene using variants from Asian-Americans (AA), Europe (E) and Africa (Af).  190,191,192,193,194,195,196,197,198 GAT HPV type 16 is a type of HPV that often infects cervical tissue which can cause cervical cancer. HPV-16 is the type that most often infects cervical tissue with prevalence almost the same as HPV Of all the mutations that occurred along the isolate sample sequence, it was found that the most common insertion was the insertion of the ATC base of all the isolate samples, it also found deletions of GAT nucleotide bases in all isolate samples, then the substitution between the purine base and other purines or the purine base with other pyrimidine bases occurred in all 100% isolate samples. In a study conducted by Gurgel et al. (2015), it is showed the occurrence of ATC insertion and GAT deletions that led to changing of amino acids in 447-threonine / 448-serine and 445-aspartate in all samples. Mutations that occur in these isolates are misense mutations which means changes that occur in nucleotides will change the amino acids produced. Changes to the HPV-16 L1 nucleotide gene can in uence capsid structure, introduction of the immune system, and neutralization of the virus (Gurgel et al., 2015).

Conclusions And Suggestions
There are molecular variations in isolates of HPV type 16 samples from combined samples in the microbiology laboratory of Andalas University, Padang, namely the insertion of ACT (6901) and Deletion (6953)  Primary ampli cation II Figure 3 Primary ampli cation III