Balkan Journal of Medical Genetics

ASSOCIATION OF VDR GENE VARIANT (rs1544410) WITH TYPE 2 DIABETES IN A PAKISTANI COHORT
Khan A1, Khan S2, Aman A1, Ali Y1, Jamal M3, Rahman B4, Ahmad M4, Aasim M4, Jalil F1,*, Shah AA4
*Corresponding Author: Dr. Fazal Jalil, Department of Biotechnology, Abdul Wali Khan University Mardan, Toru Road, Near Sheikhmaltoon Twon 23300, Mardan, Khyber-Pakhtunkhwa Province, Pakistan. E-mail: fazaljalil@awkum.edu.pk
page: 59

MATERIALS AND METHODS

Study Subjects. A total of 917 samples, which included 614 diabetes mellitus (DM) patients [469 T2DM and 145 DM type 1 (T1DM)] and 303 control samples’ data were collected from different hospitals of the District Swat, Khyber Pakhtunkhwa Province, Pakistan. A questionnaire was designed, and the patients were visited at hospitals to record various information such as random blood sugar/fasting blood sugar (RBS/FBS) tests, body mass index (BMI), age, family history and associated disease of these patients. Study participants were asked to sign a consent form provided in the questionnaire and approval was obtained from the ethics committee of the Department of Biotechnology, Abdul Wali Khan University Mardan, Marden, Khyber Pakhtunkhwa Province, Pakistan. All procedures performed in studies involving human participants were in accordance with the ethics standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethics standards. Clinical profiling of the patients was carried out by performing different clinical assessments and diagnostic tests as given below. Fasting Blood Sugar (FBS) and Random Blood Sugar (RBS) Tests. Blood samples were collected from patients in EDTA-containing vacutainer tubes and processed for RBS and FBS tests. The blood samples were centrifuged for 1 min. at 4000 rpm to extract serum. Then 10 μL serum was mixed with1000 mL glucose reagents. These test tubes were then left in a water bath for 5 min. After incubation, the mixture was put in the Microlab 330 machine (TLITech Group, Puteaux, France) to record the reading. Individuals with RBS values ranging from 70- 170 and FBS value ranging from 7-115, were considered normal, while readings higher than 170 for RBS and higher than 115 for FBS, were considered as abnormal and diabetic. The remaining blood samples were stored at –20 °C until needed for further analysis. Hb A1c Test. In this test, 100 μL Hb A1c-buffer was mixed with 5 μL blood sample in a test tube. The tubes were placed in a water bath for 12 min. These tubes were placed in the Ichroma™ machine (BodiTech Med, Inc., Chuncheon-si, Gangwond-do, Korea) to record the reading. Body Mass Index. The BMI of these patients was calculated from their body weight and height using a universal formula (BMI = weight/height). The normal BMI value ranges from 19-25. The BMI value higher than this range was considered to be obese. Glucose Tolerance Test (GTT). The GTT was done by measuring the FBS for each patient after 60, 90 and 120 min. Similarly, the sugar level in the urine of each patient was measured (Table 1). Genomic DNA Extraction and Primer Designing. Genomic DNA of 250 T2DM patients and 250 control individuals were extraverted using the organic phenolchloroform method. After extraction, DNA was quantified and then stored at –20 °C for future experiments. Genomic DNA sequence was retrieved from the National Center for Biotechnology Information (NCBI), and primers for allele-specific PCR were designed using online bioinformatics tools. Genotyping of the VDR Gene Polymorphism. For genotyping of the VDR gene variant (rs1544410), an allelespecific PCR technique was used for both case and control samples. Two forward primers, each specific to a particular allele (VDR-F1:5’-GCC ACA GAC AGG CCT GCA-3’) VDR-F2: 5’-GCC ACA GAC AGG CCT GCG-3’) and one common reverse primer (VDR-R: 5’-GTC ACT GCA CAT TGC CTC CAA-3’) was used for genotyping of VDR in the selected samples. The amplified PCR products were run on a 2.0% agarose gel and the data was noted for each allele.

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