Balkan Journal of Medical Genetics

ASSOCIATION OF RELATIVE TELOMERE LENGTH AND RISK OF HIGH HUMAN PAPILLOMAVIRUS LOAD IN CERVICAL EPITHELIAL CELLS
Albosale A H, Mashkina E V
*Corresponding Author: Dr. Abbas Hadi Albosale, Genetics Department, Southern Federal University, 344090, Stachki, 194/1, Rostov-on-Don Province, Russia. E-mail: abbashammadi4@gmail.com
page: 65

MATERIALS AND METHODS

Sample description. To conduct the molecular investigation, 100 samples were obtained from the epithelial cells scraped from the women’s urogenital tracts of the participants in the two research groups. Material for the research was obtained from the clinical diagnostic laboratory Nauka (Rostov-on-Don, Russia). Participants who were HPV-positive were subdivided into two subgroups: participants with an HPV load of 4-5 lg HPV genomes per 100 thousand human cells (n = 21), and participants with an HPV load of over 5 lg HPV genomes per 100 thousand human cells (n = 29). HPV-negative women (n = 50) were in the control group. All participants in this research project were aged 30 or over. Three inclusion criteria were applied to participants in the control group, namely: uterus negative biopsy, normal colposcopy, and HPV negative PCR testing. The case study group included women with a range of symptoms, such as abnormal menstrual bleeding, abnormal vaginal discharge, a positive uterine biopsy, and an HPV-Positive PCR-test with an HPV viral load of over 104 DNA copies per 105 human cells. Informed written consent was required from all participants before their inclusion in this research. Ethical approval was sought and secured from the Bioethics Committee of the Academy of Biology and Biotechnology of the Southern Federal University (Protocol No. 2 of March 29, 2016). Any clinical testing included in this study was conducted according to the ethical protocols issued by the World Medical Association (Helsinki Declaration). Quantitative analysis for HPV DNA. The total DNA was isolated from scrapings of epithelial cells from the cervical canal of women according to the protocols of the AmpliSens DNA-sorb-AM (InterLabService, Russia) reagent kit. The protocols specified for the AmpliSens- HPV HCR screen-titre-FRT and AmpliSens HPV HCRgenotype- FRT (Interlabservice, Russia) were employed for the genotyping and quantifying of the DNA for HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). The PCR mixture for the AmpliSens-HPV HCR screentitre- FRT contains 7 μl of PCR mix-1-FRT HPV screen titer; 8 μl mix of PCR buffer Flu and Taq polymerase and 10 μl of a DNA solution. The amplification program is as follows: 1 cycle at 95 °C for 15 minutes, followed by 1 cycle at 65 °C for 2 minutes, 93 °C for 20 seconds, 64 °C for 25 seconds, 65 °C for 55 seconds, followed by 5 cycles at 95 °C for 15 seconds, 60 °C for 25 seconds, 65 °C for 25 seconds, and 25 cycles registration. Fluorescence is measured at 60 °C in 4 channels: Fam, Joe, Rox, and Cy5 [22]. The interpretation of the viral load was performed as follows: log ≤3 per 105 human cells indicated low clinical significance, 3–5 log per 105 human cells signified clinical significance and risk of dysplasia, whereas > 5 log per 105 human cells was clinically significant with a strong risk of dysplasia, as per the guidelines and clinical reports issued by the manufacturer [22]. The PCR mixture for the AmpliSens HPV HCR-genotype-FRT contains 3.5 μl of one of the four variants of PCR-mixture-1-FRT HPV (four variants of the mixture differ in primers for specific types of HPV), 4.5 μl of a mixture of PCR-buffer-FRT and polymerase (TaqF), and 5 μl of DNA. The amplification program is as follows: 95°C for 15 minutes; 45 cycles: 95°C for 15 seconds, then 60°C for 30 seconds. Fluorescence is measured at 60 ° C [23]. Telomere and real-time polymerase chain reaction (PCR). With slight modifications, an approach was employed to ascertain telomere length according to the Cawthon method for telomere measurement by quantitative PCR [24]. The mix for the amplification reaction (25 ml) included 0.7 μl 10 mmol /L of either the telomere primers (teloF and teloR) or primers for the 36B4 gene (36B4F and 36B4R). The 36B4 gene, which functions as a reference gene that encodes acidic ribosomal phosphoprotein (PO). (Table 1) presents the primer sequence. The PCR mixture incorporated 2.5 μl 25 mM MgCl2, 2.5 μl 2, 5 ìÌ of dNTP, 2.5 μl PCR buffer, 0. 5 μl Taq-polymerase (5 U/μl), universal SYBR-Green 0.3 μl, 14 μl ddH2O (Syntol, Russia) and 2 μl DNA. Test tubes for telomeres and reference gene (36B4) (single-copy gene) were tested individually, with the findings only being calculated once an equilibrium had been established for the reaction efficiency. A real-time system (Roter-Gene, QIAGEN) was adopted for the amplification. The (36B4) thermal cycling profile included one cycle (95°C for 15 seconds) followed by 40 cycles (95°C for15 seconds, 57°C for 1 minute), while the telomere cycling included one cycle (95°C for 10 minutes) followed by 50 cycles (95°C for 15 seconds, 58°C for 1 minute). Measurement of telomere length. In the case of each DNA sample, the following calculation was made: The T/S ratio (telomere (T) was calculated for a single-copy gene (S), Telomere length was expressed as the relative T/S ratio and the relative quantification (not the absolute length) of telomeric DNA was evaluated. In each cycle, the quantity of PCR product roughly doubles. The T/S ratio was computed via the Ct (cycle threshold), which was formulated as follows: [2 Ct (telomeres)/2 Ct (36B4)]–1 = 2–ΔCt. Furthermore, the fold-change was calculated following formula: 2-ΔΔCt = 2–(ΔCt(group 1) –ΔCt (group2)) from the average of group 1 (control group) subtracted from the average of group 2 (experimental group). The data from the 2−ΔΔCt method comprises the fold change, and the analysis of the 2−ΔΔCt for the case samples compares the changes in the telomere length fold with the control samples. 2−ΔΔCt > 1 indicates that the average telomere length for the case samples is longer than that of the control sample, whereas 2−ΔΔCt < 1 suggests that the DNA samples have an average telomere length that is shorter than the control DNA samples [25]. Statistical analysis. A Pearson rank correlation coefficient (rs) was employed to evaluate the correlation between the telomere length and different parameters, including age and viral load, with a p-value less than 0.05 being deemed significant. Thus, it was possible to identify statistical differences in the telomere length in the three groups (the control group, the case study group, and the viral load group) using Student’s t-test. GraphPad InStat software (version 3.05) was utilized for all the statistical analyses.

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