Balkan Journal of Medical Genetics

GENETIC ASSOCIATION OF SOLUTE CARRIER TRANSPORTER GENE VARIANTS WITH METFORMIN RESPONSE
Abrahams-October Z1, Xhakaza L1, Pearce B1,*, Mandisa Masilela C1, Benjeddou M1, Vincent Adeniyi O2, Johnson R3,4, Jebio Ongole J5
*Corresponding Author: Brendon Pearce, Ph.D., Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa. Tel.: +2721-959-2080. Fax: +2721-959- 2648. E-mail: brendon.biff@gmail.com
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MATERIALS AND METHODS

Patients and Study Design. All participants were briefed about the project and a consent form was completed and submitted by each participant before the experiment was conducted. Ethics clearance for this study was obtained from the Senate Research Committee of the University of the Western Cape [Ethics clearance number BM/16/5/19]. Study Participants. A total of 140 T2DM outpatients belonging to the indigenous Nguni population of South Africa [Swati (n = 10), Xhosa (n = 81) and Zulu (n = 49)] were recruited from the Cecilia Makhiwane Hospital (East London, Eastern Cape) and Piet Retief Hospital (Mkhondo, Mpumalanga). Type 2 diabetes mellitus, according to the WHO criteria of 1999: plasma glucose level between 7-13 mmol/L with glycated hemoglobin (Hb) level between 7.0 and 11.0%. As some patients had other comorbidities (i.e. hypertension and dyslipidaemia) in this study, T2DM was diagnosed as a plasma glucose level between 6.0-27.0 mmol/L. Each patient participating in the study had Hb A1c levels measured within 6 month (baseline) and 12 month (follow-up) periods. Based on Hb A1c levels, patients were prescribed an average metformin dose of 1.95 mg per day (with a maximum of 2.55 mg). Patients were categorized as controlled if they demonstrated a decreased Hb A1c value less than 8.0% at 12 months in comparison to the baseline prior to the study. Uncontrolled patients demonstrated an increased Hb A1c value more than 8.0% at 12 months in comparison to the baseline prior to the study. The classification used herein for controlled and uncontrolled T2DM has been described previously [29,30]. In this pool of study subjects, 53 patients demonstrated a controlled T2DM (responders to metformin therapy), with the remaining 87 patients demonstrated an uncontrolled T2DM (non-responders to metformin therapy). Patients were included in the study if they were 18 years or older and had been on treatment for at least 1 year prior to the study. All patients were on metformin monotherapy. Patients with other diseases such as type 1 diabetes mellitus (T1DM), malignancies, hyperlipidemia, chronic kidney and liver diseases, as well as pregnant patients, were excluded from the study. Information about age, family history, medical history, demographic parameters and medication used was obtained via medical reports and interviews. In addition to this, some patients were also on antihypertensive drugs, however, while the present study does not exclude drug-drug interactions, studies have not shown that other drugs co-administered with metformin have any influence on the outcome of a genetic association with metformin response. Data Collection and Laboratory Measurements. A trained research nurse took clinical measurements of: weight, height and blood pressure (BP). Measurements were taken with all participants wearing minimal clothing and no shoes. Body mass index (BMI) for each patient was calculated as weight (kg) divided by height (m2) (Table 1). Random venous blood was collected to measure serum glycosylated Hb (Hb A1c) levels. Furthermore, lipid profile [which includes: total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and highdensity lipoprotein (HDL)] was obtained (Table 1). All blood samples were sent to relevant clinical laboratory centers for analysis. Single Nucleotide Polymorphism(s) Selection and Genotyping. The 17 relevant pharmacogenomic variants selected for this study were chosen based upon previous publications, where association was made between SNPs and response to treatment with metformin. In addition to this, variants were also cross-referenced and selected based upon an evidence level ranging between 2B-4 dictated by the pharmacogenomics knowledge base, accessed on February 5 February 2019; PharmGKB (http://www.pharm gkb.org) [31]. Genomic DNA was isolated from buccal swabs using a standard salt lysis method [32]. Samples were stored at –20 °C. DNA was quantified using a NanoDrop™2000/ 2000c UV/VIS Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The SNPs were genotyped using the MassARRAY®System IPLEX extension reaction (Agena Bioscience, San Diego, CA, USA). Genotypes of the selected SNP variants were determined for all the study participants (Table 2). Statistical Analyses. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 25 software (www.ibm.com/spss.statistics). Clinical laboratory data and anthropometric measurements were expressed as mean ± SD. Hardy-Weinberg equilibrium (HWE) p values were calculated for all SNPs using MedCalc version 2.2.0.0. (MedCalc Software, Ostend, Belgium), where p value(s) of <0.05 were considered to be significant and implied that the population was not in HWE. Association between variant(s) and response to diabetic treatment was measured using odds ratios (ORs), 95% confidence interval (95% CI) and p value(s) derived from logistic regression. The threshold for significance in association studies was set at p = 0.05.

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