The association study between changes in HbA1C with rs2250486 and rs67238751 genetic variants for SLC47A1 in newly diagnosed Iranian patients with type 2 diabetes mellitus: 6 months follow‐up study

Abstract Objectives One of the most well‐known oral medications for the treatment of T2DM is metformin. Variants have been found in studies to be useful in detecting new genes connected to T2DM aetiology and affecting metformin's mechanism of action. In this research, we aimed to study two variations of the SLC47A1 gene; rs2250486 and rs67238751, in T2DM patients who had been taking metformin for the first 6 months after the diagnosis in the Iranian population for the first time. Design and Methods A total of 200 individuals were recruited for the study. According to their glycosylated haemoglobin (HbA1c) levels, the patients were divided into two groups: responders (HbA1c levels were reduced by at least 1% after 6 months of metformin treatment.) and non‐responders. DNA was extracted from whole blood and genotyped by Tetra ARMS PCR. High‐performance liquid chromatography (HPLC) was used to measure HbA1c levels at the start of the treatment and again 6 months later. Results rs2250486 variant in the dominant model reduces the HbA1C levels after 6 months of metformin treatment. In fact, when compared to the T/C + C/C genotypes, the T/T genotype improves HbA1C levels (p‐value = .014). Furthermore, in the allelic model, the T allele improves HbA1C levels in comparison to the C allele (p‐value = .008). After 6 months of metformin treatment, serum levels of HbA1C in responders were reduced significantly in both groups (T/T and T/C + C/C), (p‐value = <.0001). However, the rs67238751 variant did not reveal a meaningful relationship with lower HbA1C levels in any of the models. Conclusions This study found that the rs2250486 variant could be associated with reducing HbA1C levels while the rs67238751 variant, had no relationship.


| INTRODUC TI ON
Type 2 diabetes mellitus (T2DM) is one of the most common metabolic disorders worldwide. Deficiency in insulin release by pancreatic beta cells and decreased tissue sensitivity to insulin are the two main causes of the disease. The insulin synthesis and molecular secretion mechanisms, as well as the insulin response in target tissues, must be ideally co-ordinated. As a result, a defect in the involved mechanisms can lead to a metabolic imbalance, which can contribute to the pathogenesis of T2DM. 1,2 Having a genetic background plays an important role in T2DM. The polygenic nature of the disease has been discovered in studies during the last decade. The majority of these genes have the ability to influence insulin secretion and its activity. 3 One of the most frequent medications used to treat T2DM is metformin, which is made from biguanide. Metformin can be used single or in combination with other medications like insulin to treat diabetes. 4 Metformin is actively transported through organic cation transporters (OCTs) in enterocytes, liver cells, and renal epithelial cells. Multidrug and toxin extrusion transporter 1 (MATE1), encoded by SLC47A1 transports metformin from liver to the bloodstream. Finally, metformin is secreted into the urine via MATE1 and MATE2 (encoded by SLC47A2) 5,6 (Figure 1). On a single-medication treatment with metformin, over 35% of patients with T2DM fail to regulate their serum blood glucose concentrations. 7 The relationship between genetic variations of metformin transporters and treatment results has been discovered in a number of studies. 8 Genetic variations can affect gene expression in a variety of ways, resulting in changes in treatment responsiveness. 9 Expression quantitative trait loci (eQTLs) are defined as genetic variations that are linked to gene expression and are available at the genotype-tissue expression (GTex) portal. 10 Variations in the SLC47A1 gene, which encodes one of the metformin kidney transporters, might have a significant impact on metformin reabsorption via MATE1. However, only a few contradictory studies on the effect of the SLC47A1 gene on metformin efficiency have been conducted around the world. 11 The current study focuses on two intronic variants in the SLC47A1 gene, rs2250486 (T > C) and rs67238751 (C > T), which might have an impact on metformin responsiveness and reducing HbA1C levels in T2DM patients in the Iranian population for the first time.
This study evaluated the effects of these variants on HbA1c levels and other biochemical and anthropometric parameters like systolic and diastolic blood pressure (SBP, DBP), lipid profile, fast blood sugar (FBS) and body mass index (BMI) before and after metformin treatment in both responder and non-responder groups. It also looked at the effect of dominant, co-dominant and recessive models in the genotypic groups with the effect of metformin treatment. Clinic and newly diagnosed with T2DM based on world health organization (WHO) recommendation. 13 Patients with liver disorders such as hepatitis, infectious and inflammatory diseases, kidney and autoimmune diseases, and pregnant women were excluded from the trial, as were those who received insulin or other oral antidiabetic medications. The number of people in the group who responded to treatment was 102 (The reduction in HbA1C levels was more than 1% after 6 months of monotherapy with metformin). Whereas the number of people who did not respond to metformin treatment was equal to 98.

| Blood sampling
A questionnaire was used to collect information such as age, sex and history of endocrine, hepatic, infectious, and inflammatory disorders, as well as a history of blood-sugar-lowering medications and the length of diabetes. Biochemical and anthropometric parameters were analysed at baseline and after 6 months of monotherapy. Blood sampling was performed after 10-12 h of fasting. FBS, triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by the HITACHI auto-analyser. sdLDL was calculated through the TG/HDL formula. HbA1c levels were analysed by the HPLC.
Individuals' weight and height were measured using standard procedures, and BMI was calculated using the standard formula (kg/m 2 ).
Blood pressure was measured in a sitting position and after 15 min of rest using a standard blood pressure monitor.

| Treatment protocol
We enrolled 200 Iranian patients who were newly diagnosed with T2DM, and all patients were only treated with metformin for the first 6 months after the diagnosis. An identical dose (1000 mg/day) was given to all of the participants. All patients were instructed to follow a low-carb diet under the guidance of a dietician after the initial diagnosis and the beginning of treatment, and to walk on average 3 days per week.

| Genotyping
Three millilitre of patients' blood was stored at −80°C in vials containing ethylenediaminetetraacetic acid (EDTA) until the day of DNA extraction. The salting-out approach, which is a quick and cost-benefit method for DNA extraction, was applied in this work.
The concentration of extracted DNA, as well as the purity of the samples, were determined using a Nano-drop spectrophotometer.
For each variant, Tetra ARMS PCR was used for genotyping. The thermocycler setup programs for the variants are shown in Table 1.
After the PCR, the products were evaluated using a 2% agarose gel electrophoresis. For primer design, the primer1 program was

| In silico analysis
The PharmGKB (https://www.pharm gkb.org/) database was used to find metformin-related genes, and after selecting the SLC47A1

| Statistical analysis
PGA software was used to calculate the sample size. 14 In this study, the abundance of alleles and genotypes and Hardy-Weinberg equilibrium were investigated using SNPStats software (https://www. snpst ats.net/). Patients' data were analysed using SPSS software (version 25). Descriptive statistics in the present study are expressed as the average ± SD. The Shapiro-Wilk test was used to determine the normality of the data distribution. Also, a 2-way ANOVA test was used to compare parametric variables.

| Patients' characteristics
The study included 200 patients, 97 men and 103 women, and found that 102 patients responded to metformin treatment, and 98 patients did not. There were 48 women and 54 men among the responders.
There were 57 women and 41 men among the non-responders. The average age ranged from 55.84 ± 4.8 in the responder group and 56.99 ± 4.85 in the non-responder group. The difference between these two groups was not significant (p-value = .9). The Mann-Whitney U test was utilized due to the anomalous age distribution in both groups. Table 3 shows the genotypes and allele distributions of the studied variations. The two variations have a significant correlation (D′ = 0.307, r 2 = 0.073) according to online SNPSTATs software.

| rs2250486 (T > C)
According to data analysis, the distribution of genotypes in the two groups of responders and non-responders is in Hardy-Weinberg equilibrium (p-value = .295 and .367 respectively).

| rs67238751 (C > T)
According to  Table 3 shows the changes in biochemical and anthropometric parameters in the responder and non-responder groups according to T/T genotype and C allele carriers of rs2250486, after 6 months of TA B L E 2 Genotypes and allele frequencies of rs2250486 and rs67238751 in responder and non-responder groups. .5145

| Comparison of anthropometric and biochemical parameters in variant rs2250486
Recessive   metformin monotherapy. In the responder group, the BMI parameter in both T/T genotype and C allele carriers was not significantly reduced (p-value = .541 and .5144 respectively). After 6 months of treatment, SBP declined significantly in both groups, although it was even lower in individuals with the T/T genotype (p-value = .0001).

TA B L E 3 Comparison of anthropometric and biochemical parameters before and after treatment in T/T and T/C + C/C groups in
In T/T genotype responders, the DBP parameter fell significantly (p-value = .0004), but not in C allele participants (p-value = .3144).
After 6 months of metformin treatment, serum levels of TC, HbA1C and FBS dropped significantly in both groups (p-value = <.0001, <.0001, <.0001 respectively). In addition, the average HDL-C in both the C allele carriers and T/T genotype groups was not substantially lower than before the treatment (p-value = .3065 and .576 respectively). The C allele carriers group had a substantial reduction in TG parameter (p-value = .0077). However, the T/T genotype group had no significant drop (p-value = .2787). And finally, the LDL-C parameter in T/T genotype group demonstrates a significant decrease (P-value = .0229). sdLDL showed a significant decrease in T/T and T/C + C/C genotypes only in responder group (p-value = .0423, .0067 respectively). However, in the non-responder groups, Individuals with the T/T genotype did not exhibit a significant reduction in the investigated parameters after 6 months of metformin treatment except for HbA1C levels that dropped considerably (p-value = .007).
Only DBP and HbA1C levels were significantly lower in C allele carriers (p-value = .0279 and .010 respectively). However, in general the drop in HbA1C in these patients was insufficient to put them in the responder group (reduction was <1%). Table 4 represents a comparison of the examined parameters in the responder and non-responder groups with the T allele carriers and the C/C genotype before and after 6 months of metformin monotherapy. The mean BMI, HDL-C and TG parameters with the C/C genotype in responder group did not appear to be reduced in this table (p-value = .4349, .1202 and .1047 respectively). The SBP parameter drops in both T allele carriers (p-value = .0128) and C/C genotype, but the latter group's decline is considerably more substantial (p-value = <.0001). The DBP parameter was considerably lower in people with the C/C genotype (p-value = .0015), but not in people with the T allele carriers (p-value = .0956). The T allele carriers have significantly lower mean TC, FBS and HbA1C values (p-value = .006, <.0001 and <.0001 respectively). In the C/C genotype group, this drop is likewise significant (p-value = <.0001, <.0001 and <.0001 respectively). It is worth to mention that, sdLDL parameters in C/C and C/T + T/T in responder group had a meaningful reduction (p-value = .0273, .272 respectively). Finally, in the C/C genotype group, the LDL-C parameter was considerably reduced (p-value = .0356), whereas this was not detected in the T allele group (p-value =.1834). As shown in Table 4, in the nonresponder group with the T allele carriers and the C/C genotype, except for the HbA1C (p-value = .0001), there was no significant reduction in any of the parameters before and after metformin treatment. Moreover, the reduction in HbA1C levels in these people was not significant enough to put them in the responder group (reduction was <1%). Additionally, the groups interaction on TC is meaningful either way (p-value = <.0001). Despite the effect of groups on SBP, there was no evidence of interaction (p-value = .12764).

| DISCUSS ION
Type 2 diabetes is a metabolic disease that manifests as a chronic disease characterized by elevated blood glucose concentration.
Because it raises the risk of heart disease, stroke, peripheral neuropathy, renal disease and amputation, and is linked to a shorter life expectancy. 15 Diabetes has quadrupled in global prevalence during the previous three decades, and it is now the ninth greatest cause of mortality. Asia is a significant hotspot for the global T2DM epidemic, which is quickly growing. 16 The most frequent treatment for T2DM is metformin, which reduces blood glucose concentrations in these patients. In recent years, studies have found that this medication has a remarkable effect on cardiovascular disease, neurological ailments and polycystic ovarian syndrome. 17 According to clinical trials, more than a third of those taking metformin as a single dose do not achieve low fasting glucose levels.
Changes in genes involved in the pharmacokinetics and pharmacodynamics of metformin could be the major reason for these people's lack of responsiveness to this medication. 18   increase disease risk or alter the connection between genotype and phenotype. However, some intronic polymorphic variants can still affect the splicing phenotype by being found within an intron splice enhancer or branchpoint site, or by activating a cryp-  SLC47A1 gene will provide more information on the efficacy of metformin in T2DM patients.

| CON CLUS ION
It seems that the rs2250486 variant might be an important factor in reducing HbA1C concentration in T2DM patients in the Iranian population, and in contrast to rs67238751 in response to metformin, no relationship was found. However, a larger sample size is required to complete this result.

ACK N O WLE D G E M ENTS
We appreciate Dr Hamid Ghaedi as the Medical genetic specialist for this research for his generous co-operation and his helpful consultations.

FU N D I N G I N FO R M ATI O N
The present article is financially supported by 'Research department of the school of Medicine-Shahid Beheshti University of Medical Sciences' (Grant No: 24139).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no relevant financial or non-financial interests to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be made available on reasonable request.

E TH I C A L A PPROVA L
This study is performed in line with the principles of the Declarations

CO N S E NT TO PA RTI CI PATE
Informed consent was obtained from all individual participants included in the study.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.