155 Effects of SLC22A2 (rs201919874) and SLC47A2 (rs138244461) genetic variants on Metformin Pharmacokinetics in Pakistani T2DM patients Sadaf Moeez,1 Zoya Khalid,2 Fazal Jalil,3 Muhammad Irfan,4 Muhammad Ismail,5 Mohammad Ali Arif,6 Rauf Niazi,7 Sumbul Khalid8 Abstract Objective: To determine the frequencies of single nucleotide polymorphisms rs201919874 and rs138244461 in genes SLC22A2 and SLC47A2 respectively in Pakistani diabetes patients in order to characterise the genetic variants and determine their association with the pharmacokinetics of metformin. Methods: The case-control study was conducted at the International Islamic University, Islamabad, Pakistan, from June 2016 to June 2017, and comprised genotypes of diabetic cases and matching controls which were determined following allele-specific polymerase chain reaction. Cases were further divided into Group A and Group B. The former consisted of diabetics who were on monotherapy of metformin, while the latter consisted of diabetics treated with a combination of metformin and sulfonylureas. In-silico analysis was performed to verify the effect of single nucleotide polymorphisms rs201919874 and rs138244461 on the structure of genes. Association was statistically determined using SPSS 18. Results: Of the 1200 subjects, 800(66.6%) were cases and 400(33.3%) were controls. Among the cases, 400(50%) each were in Group A and Group B. Significant difference was observed in the distribution of rs201919874 between Group A and controls (p<0.05) and between Group B and controls (p<0.05) for heterozygous genotypic frequency and for allelic frequency. Conversely, statistically significant difference was observed in rs138244461 (p<0.05) for all genotypic and allelic frequencies. Genotypes were significantly associated with glycated haemoglobin, random and fasting glucose levels in Group A compared to Group B (p<0.05). In-silico analysis showed that both single nucleotide polymorphisms were expected to create significantly damaging structural changes in domains and helix (p<0.05 each). Conclusions: Both exonic single nucleotide polymorphisms were found to be associated with the pharmacokinetics of metformin. Key Words: Diabetes, Metformin, Pakistan, SNPs. (JPMA 69: 155; 2019). Introduction not undergo under any kind of metabolism by hepatic Metformin has been most commonly used as a first-line enzymes and is excreted unchanged by the kidneys. therapy for treatment of type 2 diabetes mellitus (T2DM) Transporters of metformin play a key role in its distribution for decades due to both its good anti-hyperglycaemic to different tissues and in its elimination through renal effect and safety profile.1 The pharmacological basis of passage.3 A considerable inter-individual variability in how metformin lowers the glucose level is not completely glucose-lowering response to metformin was reported clarified, but it has been established that its key function previously along with reduction of glycated haemoglobin is to inhibit hepatic gluconeogenesis.2 Metformin does (HbA1c) values ranging from 0.8% to 3%. Furthermore, 1,8International Islamic University, Islamabad, 2Sabanci University, Istanbul, less than two-thirds of patients responded adequately to 3Abdul Wali Khan University Mardan, 4Pir Mehr Ali Shah, Arid Agriculture metformin and achieved a desired fasting blood sugar University, Rawalpindi, 5Institute of Biomedical and Genetic Engineering, (FBS) level.4-7 Islamabad, 6,7Pakistan Institute of Medical Sciences (PIMS), Islamabad Correspondence: Sumbul Khalid. e-mail: [email protected] Human organic cation transporters (OCT 1-3) are encoded J Pak Med Assoc Effects of SLC22A2 (rs201919874) and SLC47A2 (rs138244461) genetic variants..... 156 by genes SLC22A1, SLC22A2 and SLC22A3. These are poly- kidney and its elimination from the organ. Hence, the specific transporters for small and hydrophilic organic present study was planned to evaluate the occurrence of cations like endogenous compounds serotonin and SLC22A2 (rs201919874G>A) and SLC47A2 (rs138244461 dopamine, toxic substances and clinically used drugs. C>T) genetic polymorphisms in T2DM patients that were Among more than 120 clinically used drugs that interact on monotherapy of metformin and those taking with different human OCTs, at least 20 are well-known combination therapy along with sulfonylureas. being transported. These include the anti-diabetic drug metformin, antineoplastic platinum compounds, the Material and Methods antiviral drugs acyclovir, the histamine H2 receptor The case-control study was conducted at the International antagonist cimetidine, ganciclovir, lamivudine and Islamic University, Islamabad, Pakistan, from June 2016 zalcitabine, and the antiarrhythmic drug quinidine.8 to June 2017, and comprised genotypes of diabetic cases All solute carrier superfamily (SLC22) proteins share a and matching controls which were determined following common membrane topology with 12 -helical allele-specific polymerase chain reaction. Cases were transmembrane domains. Mutational analysis and further divided into Group A and Group B. The former homology modelling of the steric structure of the proteins consisted of diabetics who were on monotherapy of led to the conclusion that they possess a large cleft that metformin, while the latter consisted of diabetics treated is accessible from the aqueous phase. Located within this with a combination of metformin and sulfonylureas. 20 cleft is an inner cavity containing different interaction Sample size was calculated by using online calculator sites for different substrates.9 It is involved in the uptake by considering confidence level 95% and confidence 21 of various xenobiotics from the bloodstream and takes interval (CI) in line with literature. Patients in group A them into renal epithelial cells.10 Kimura et al used Human were taking 1500mg of metformin per day for 6 months embryonic kidney (HEK293) cells to check the expression or more, while those in Group B was on 1000mg of of OCT2 and illustrated that metformin is a good substrate metformin and 80mg of sulfonylureas per day for one for this transporter.11 Different functional variants have year or more. Fom each individual, 3-5 ml blood sample been identified in the gene SLC22A2 that encodes OCT2 was taken and collected in ethylenediaminetetraacetic transporter.12 acid (EDTA) tubes. Multidrug and toxin extrusion (MATE) transporters are Cases were defined as subjects of either gender aged 35- encoded by genes SLC47A1 and SLC47A2. They are involved 80 years. Diagnosis of T2DM was based on the World in the efflux of several lipophobic organic cations, Health Organisation (WHO) / American Diabetes 22,23 including metformin. These transporters contain 400 to Association (ADA) definition. Patients with other types 550 amino acid residues and span 12 transmembrane of diabetes, co-treatment with other anti-diabetic drugs, domains.13,14 Genes of these transporters are located on and pregnant women were excluded. Written informed the short arm of the 17th chromosome, 17p11.2.15 Two consent was taken from all individuals prior to the study isoforms of MATE2 have been identified one of which is which was approved by the Pakistan Institute of Medical MATE2K.16 Like MATE1, MATE2K has been involved in the Sciences (PIMS) Hospital, Islamabad, Pakistan. Blood transport of several structurally distinct compounds, sampling from T2DM patients was done in outpatient including metformin.17 Up till now, only few genetic clinics of endocrinology at PIMS. Detailed demographic variants have been identified in MATE2K and very few and clinical data was collected from each individual. have been analysed with respect to metformin.18 Unrelated healthy volunteers of either gender were enrolled through non-probability consecutive sampling. Genetic variation in the genes SLC22A2 and SLC47A2 that encodes OCT2 and MATE2K transporters have been found Genomic deoxyribonucleic acid (DNA) was isolated from to be linked with therapeutic efficacy of metformin in peripheral blood leukocytes using standard phenol O T2DM.10,19 Both single nucleotide polymorphisms (SNPs) chloroform method and stored at -20 C, until use. are present in exon so it was hypothesized that any Genotyping was done using allele-specific polymerase changes in these sequences may reduce transcription chain reaction (PCR) and amplification was done by using rates and thereby reduced OCT2 and MATE2K expression 2700 Applied Biosystems. The primers sets, two forward leading to a decreased transport of metformin into the primers and one reverse primer, were used to amplify Vol. 69, No. 02, February 2019 157 S. Moeez, Z. Khalid, F. Jalil, et al. ACLY Median ranges were used to describe the central tendency SLC22A1 SLC47A2 ALDH18A1 and variability of continuous variables, while frequencies ZNF200 EHMT2 TOP2B TOP2A were used to describe the distribution of categorical SLC22A6 variables. Fisher exact test or non-parametric Mann- YRDC IGF2R SLC22A8 Whitney test was used to compare clinical characteristics RS1A1 SLC22A2 SLC47A2 SLC6A2 SLC22A2 between different patient groups. Chi-square test was SLC2A12 used to assess the deviation from Hardy-Weinberg SLC22A13 equilibrium (HWE). The level of statistical significance was SLC2A13 SLC47A1 set at p<0.05. Data was analysed using SPSS 18. SLC22A5 Results Figure-1: Gene network of SLC22A2 and SLC47A2 predicted by STRING. Of the 1200 subjects, 800(66.6%) were cases and 400(33.3%) were controls. Among the cases, 400(50%) SLC22A2 rs201919874 and SLC47A2 rs138244461