Study Site:
The Coimbatore district lies between 11.0168°N and 76.9558°E latitude and longitude. It is one of the most attractive, beautiful and modern cities of the south region of India. Chennai district lies within 13.0827°N and 80.2707°E. It is one of the most populous cities of India and the Capital of Tamil Nadu state located in southern most part of Indian peninsula. Nilgiris district is lying in the 11.4916°N and 76.7337°E latitude and longitude. It is one of the smallest district in the Tamil Nadu which contains a range of mountains where the climate is always cold. Dharmapuri is a town that lies in the western past of Tamil Nadu which has comes under the latitude and longitude of 12.1211°N and 78.1582°E. From these districts the samples were categorised as urban (n=46), rural (n=18) and tribal (n=08). The study data was categorized based on age as group I (<55) and group II (≥55). Based on the categorization, the urban group I consists of n=18 and group II, n=28, in rural group I, n=06 and in group II, n=12, whereas in tribes group I comprised of n=03 and group II, n=05.
Study population:
Totally 72 OC patients were selected from urban (n = 46), rural (n =18) and tribal (n=08) populations and the blood samples and questionnaire was obtained from various oncology clinic, medical hospitals and population-based survey from Coimbatore, Chennai, Dharmapuri and Nilgiris districts, South India. The control group was also selected according to the age matched and population residing region.
Study design:
Institutional Human Ethical clearance was obtained from “The Avinashilingam University for Women, Coimbatore (Ref No. AUW/IHEC-17-18/ZOO/FHP-10), and Balaji Medical College – Chennai (Ref No. 002/SBMCIHEC/2017/986). The Helsinki (1966) declaration was followed through-out the study. Informed consent had been obtained from the study subjects. A brief flow chart describes the study design which has been depicted in fig. 5.
Inclusion criteria:
The following categories were considered as the prime inclusion criteria among the OC subjects for the present study includes age, type I and II OC, stage I and II of OC, OC patients undergone chemotherapy and surgery, education qualification, parity status and lifestyle (IVF, medications and menarche).
Exclusion criteria:
The patients who were bed-ridden, immunological diseases, Gestational women, or any other genetic disorders were excluded from the present study. OC patients who had undergone radiation therapy were also excluded from this study.
The survey questionnaire:
The survey questionnaire was aimed at screening the patients with OC in three different populations including urban, rural and tribes. The survey consisted of three sections. Section 1 focused on demographic details like age, education, food habit, and parity status. Section 2 consisted of questions about specific characteristics observed in OC patients, which included the age of menarche, intake of natural or oral contraceptive medicines and pregnancy attained due to IVF. Section 3 concentrated on symptoms of OC in patients, particularly abdominal pain or pelvic pain, sudden weight loss, frequent urination, indigestion and abdominal bloating. A team of oncologist and gynaecologist had developed the questionnaire and it covered most of the common symptoms found in OC. Language experts translated the questionnaire from English to Tamil.
Blood Sample Collection:
Blood Sample Collection: Peripheral blood of about 10 mL was collected from the individuals through venipuncture and were stored in anticoagulants tubes. After collection, minimal amount of serum was harvested in Eppendorf tube and stored at -80°C until laboratory use.
Hormonal Assay:
Dispense 50 μl of standard, specimens, and controls into appropriate wells. Dispense 100 μl of Enzyme Conjugate Reagent into each well. Gently mix for 30 seconds. Incubate at room temperature (18-25°C) for 45 minutes. Dispense 100 μl TMB Reagent into each well. Gently mix for 10 seconds. Incubate at room temperature in the dark for 20 minutes. Stop the reaction by adding 100 μl of Stop Solution to each well. Gently mix for 30 seconds. Read the optical density at 450 nm with a microtiter plate reader within 15 minutes. The protocol was conducted using the Abcam hormonal kit and the protocol was followed according to the manufacturer’s instruction.
MT-DNA isolation from blood:
The mtDNA protocol was conducted according to the Thapa et al.,31. The postnuclear supernatant fraction was collected in the 30 mL centrifuge tube and centrifuged at 15000 g at 4°C for 30 min to sediment the mitochondrial pellet. Wash the pellet thrice with low salt buffer (TKM1), to ensure complete elimination of lysed erythrocytes. Then the pellet was transferred to a 2 mL Eppendorf tube and suspended in 480 μL of high salt buffer TKM2followed by addition of 75 μL of 10% SDS. Then it was incubated at 55°C in the heating block for complete denaturation and solubilization of protein. After that 200 μL of 6 M NaCl was added followed by centrifugation at 11300 g for 20 min in the microcentrifuge. Subsequently, the supernatant was transferred to a 2 mL tube. Then twice the volume of 100% ethanol was added for complete precipitation of mtDNA pellet followed by washing the pellet twice with 70% ethanol. It was then dried and finally dissolved in 200 μL of sterile water. The concentration of DNA was assessed by taking nanodrop readings at 260-280nm, checking the quality of DNA by agarose gel electrophoresis.
PCR amplification of MT-ATP6 gene:
The MT-ATP6 gene was amplified by PCR and ATPase region was carried out in 25 μl total reaction volume, each containing 100 ng of template DNA, 0.25 pM of each primer, 2.5 μl of 10X PCR buffer, 1.5 mM MgCl2, 200 mM dNTPs, and 1.5 U of Dream Taq green DNA polymerase. PCR volume was heated initially to 95 °C for 5 mins followed by 30 cycles each consisting of 1 min denaturation at 95 °C, 40 s annealing at 56 °C, 1 min extension at 72 °C. The reaction ended with a final extension step by incubating at 72 °C for 5 min. The PCR products were subjected to electrophoresis in a 1.2 % Agarose gel in 1X TBE buffer, stained with Ethidium Bromide (EtBr).
Sequencing Analysis:
All PCR products were sequenced using the previously reported primers. The sequence analysis were compared to the human mtDNA reference sequence using the BLAST sequence analysis tool. Mitochondrial genome sequence variations were identified using the Mitomap database. Each polymorphisms obtained were then verified against the Mitomap database. Sequence variants that were not found in that database were classified as new polymorphism/mutation, whereas others already reported were classified as reported polymorphism/mutation.
Data Analysis:
Statistical analysis was performed using SPSS 13.0 Windows Software. Mean and Standard Deviation were used for continuous variables. To quantify the correlation between survival time and each independent feature, a 95% confidence interval (CI) was used. All values were considered to be significant at p <0.05 level throughout the study.