In this case-control study all the volunteers had to follow a careful screening program. A total of 603 patients recruited from OPD Medicine, King George’s Medical University, Lucknow, India. 20 subjects were omitted from the study due to their personnel reasons and not willing to give consent. Lastly, 300 T2DM subjects and 300 control subjects were employed for this study. The subjects were general population from Uttar Pradesh, India. T2DM is defined as fasting plasma glucose level FBS ≥ 126 and HbA1C (≥ 6.5%) level repeatedly. This was defined according to the American diabetes association [7]. Additionally, subjects with a diagnosis of T2DM and a history of at least 2 years of treatment without insulin use were enlisted. Controls were healthy group and cohort of KGMU employees and had no familial history of T2DM. Participants identified with T1DM, heart disease, liver cirrhosis, renal disease gastrointestinal disease, pulmonary disease or cancer were debarred for adjustment of confounding factors. Participants were interviewed using a questionnaire for collecting demographic and laboratory investigations. This study was approved by institutional ethics committee, KGMU. approved the ethical consent. All subject signed the approved informed consent.
Blood sample collection
Total of 3mL of venous blood was withdrawn from each participant into anticoagulant EDTA vials (BD Vacutainer®) for extraction of genomic DNA. The DNA was then stored in TE buffer and divided into aliquots then stored at -20° C for genetic analysis. Further, 2mL of whole blood was centrifuged at 1,500 rpm for 10 min and then separated serum was used for the analysis of biochemical profile and HOMA-IR.
Insulin resistance calculation
Insulin sensitivity was assessed by calculating homeostasis Model Assessment parameter of Insulin Resistance
HOMA = insulin(mU/m) * [glucose/22.5]
QUICKI = 1/ (log Fasting Insulin + log Fasting Glucose in mg/dL)
Patients were considered as insulin resistance when HOMA ≥ 2.6 (high value indicate insulin resistance) and QUICKI ≤ 0.33 (low value reflects insulin resistance). Fasting insulin was considered to evaluate IR [8].
DNA amplification and Genotyping
DNA was isolated from freshly drawn blood (leucocytes) gained from EDTA tubes by using salting out method. The SCARB1 gene were amplified by polymerase chain reaction using specific primer designed by using Primer3. Further, the PCR products amplified to identify the genotypes were incubated with specific restriction enzyme as recommended by the manufacturer’s protocol. Genotypes were determined after electrophoresis on agarose gel stained with 0.5 µg/ml ethidium bromide visualized in ultraviolet light.
SCARB1 rs74830677
SCARB1 gene polymorphism was detected by polymerase chain reaction (PCR; Applied Biosystems™ Veriti™) followed by restriction fragment length polymorphism. SCARB1 gene was amplified by the following PCR conditions: 92° C for 6 minutes, followed by 36 cycles of 92° C for 33 seconds, 61° C for 35 seconds, 68° C for 35 seconds and final elongation at 73° C for 6 minutes with precise SCARB1 rs74830677 gene forward primer 5’-TGCTCCAACCAGGAATCAC-3’ and reverse primer 5’-CCATCCTCACTTCCTCAAC-3’ (Thermoscientific). Amplification was done with a 13 µL PCR mixture comprising 0.5 µL template DNA, 0.5 µL of both primers and master mixes (Thermo Fisher Scientific Inc.). Enzyme digestion was directed in a 13µL final volume comprising of 1 unit of the SetI enzyme. The reaction was conducted at 37° C overnight and the digested products were separated on 2% agarose gel electrophoresis containing EtBr. The genotypes recognised were categorized according to the presence or absence of the enzyme restriction sites. As a result, the TT genotype was a wild homozygote, the CT genotype was a heterozygote and the CC genotype was a variant homozygote.
Expression analysis of SCARB1
The SCARB1 mRNA levels were assessed in 125 Healthy controls and 125 with T2DM. Isolation of total RNA from peripheral blood was performed by using the mini kit (Qiagen, Valencia, CA). Total RNA (10µg) was converted to cDNA using high-capacity cDNA reverse transcription kit (Catalog number: 4368814, Applied Biosystems™). Real-time PCR was performed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using βeta actin gene mRNA was used as a reference standard. Reaction was performed by Initial denaturation for 2 min at 50°C and for 10 min at 95°C, followed by 40 cycles of PCR (95°C for 15 s; 60°C for 1 min). Reactions were performed using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). CT method for the relative expression of results were used for the comparison between genes in T2DM and healthy controls.
Statistical analysis
The differences of all parametric variables were analysed by using student’s t test. Chi square (χ2) test was applied to determine the significance of differences in allele and genotype frequency. Allele and genotype frequencies for SCARB1 were calculated by gene counting method. Analysis of variance (ANOVA) was used to test for variance in parameters between genotypes. Differences were considered significant when p was < 0.05. All statistical analyses were performed u sing SPSS software version 16.0 (SPSS, Chicago, IL).