2.1. Study design and subjects
A total of 100 Egyptians adult subjects was divided into 2 groups 50 with MetS (25 males and 25 females) and 50 healthy control subjects (12 males and 38 females). Samples were collected from local, public, and private hospitals after obtaining informed consent according to WHO instructions. The study was carried out at National Research Centre of Egypt. The study protocol was approved by the ethics committee board of the Ministry of Health and Population in Egypt (No: 23-2019/20). In the present study MetS was diagnosed by the presence of three or more of the following risk factors according to the International Diabetes Federation (IDF) : waist circumference ≥94 cm in men and ≥80 cm in women, serum triglycerides (TG) ≥150 mg/dL; high-density lipoprotein cholesterol (HDL-C) ≤40 mg/dL in men and ≤50 mg/dL in women, blood pressure (BP) ≥130≥85 mmHg; or fasting plasma glucose (FPG) ≥ 5.6 mmol/L.
2.2. Anthropometric parameters and Biochemical analysis
Anthropometric measurements were obtained according to standardized equipment and following the recommendations of the International Biological Program. Body weight, height, waist and hip circumferences were measured. All measurements were taken 3 times on the left side of the body, and the mean of the 3 values was used. Body weight was measured with the patients in light clothing and without shoes. Height was measured with the patients standing with their backs leaning against the stadiometer of the same scale. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters square (kg/m2). Waist circumference (WC) and hip circumference (HC) were measured in cm using a plastic, non-stretchable tape. WC was measured with light clothing at a level midway between the lower rib margin and the iliac crest standing and breathing normally, Waist to hip ratio (WHR) was calculated. Blood pressure was measured by the auscultatory method after the subject had been sitting at rest for a minimum period of 5 min, and the cuff involved 80% of the right arm circumference. The arm rested on a support surface at the level of the precordium. Blood pressure was measured three times and was averaged for analysis.
Venous blood samples were collected by direct venipuncture after an overnight fast. Fasting plasma glucose and serum lipids (total cholesterol, high-density lipoprotein cholesterol (HDL-C) triglycerides (TG) were measured by enzymatic colorimetric methods using a Hitachi autoanalyzer 704 (Roche Diagnostics. Switzerland). Low density lipoprotein cholesterol (LDL-C) was calculated according to certain equation (LDL-C= Total cholesterol –Triglycerides/5+ HDL-C) .
2.3. RNA extraction and quantitative real-time PCR
RNA was extracted using RNeasy kit of Qiagen (Germany) according to the manufacturer’s instructions. For RNA reverse transcription, RNA was reverse transcribed to cDNA using RNA Reverse Transcription Kit (Applied Biosystems) and random primer according to the manufacturer’s instructions. Reverse transcription was performed under the following conditions: 2 hrs at 37°C, 20 min at 85°C, then the resulting cDNA was kept at -80°C until use. A real-time quantitative PCR (qRT-PCR) was carried out to quantify the expression levels in triplicate of RNA using Taqman® RNA Assay kit (Applied Biosystems) and Taqman® Universal Master Mix (Applied Biosystems) using step one real time PCR system (Applied Biosystems) according to the manufacturer’s instructions. GAPDH (Applied Biosystems) was used as endogenous control to normalize the expression levels of DYRK1B gene. The qRT-PCR protocol was as follows: initial denaturation at 94°C for 20s followed by annealing at 56°C for 20s and extension at 72°C for 30s for 45 cycles. Relative quantification (Rq) of DYRK1B expression was calculated using the 2-ΔΔCT threshold cycle method. ΔCt was determined by subtracting the Ct values for GAPDH from the Ct values for the DYRK1B gene.
2.4. Statistical analysis
Data were statistically described in terms of mean ± standard deviation (±SD), median and range, or frequencies (number of cases) and percentages when appropriate. Numerical data were tested for the normal assumption using . Comparison of numerical variables between the study groups was done using Student t test for independent samples. For comparing gender, Chi-square test was performed. Two-sided p values less than 0.05 was considered statistically significant. All statistical calculations were done using computer program IBM SPSS (Statistical Package for the Social Science; IBM Corp, Armonk, NY, USA) release 22 for Microsoft Windows.