Integrated analysis of gene expression of solute carrier gene SLC19A3 with proven risk factors of microvascular complications in patients with type 2 diabetes

Integrated analysis of gene expression of solute carrier gene SLC19A3 with proven risk factors of microvascular complications in patients with type 2 diabetes K. Singh, T. Yu. Yuzvenko Ukrainian Research and Practical Centre for Endocrine Surgery and Transplantation of Endocrine Organs and Tissues, MOH of Ukraine, Kyiv Diabetes mellitus (DM) is a chronic progressive disorder which leads to significant disability, morbidity and is likely to progress to become one of the most widespread conditions worldwide and as an additional burden to the healthcare system already reeling under the effects of the COVID-19 pandemic worldwide. Aim — to study the correlative variation in gene expression of SLC19A3 in type 2 diabetes patients with proven risk factors of diabetes complications. Materials and methods. In the study, 190 patients with type 2 DM were screened for diabetic peripheral neuropathy (DPN). DPN was confirmed in (n = 105) patients displaying symptoms of diabetic polyneuropathy with the involvement of small nerve fibers and large nerve fibers. Out of the total cohort, 45 patients with type 2 DM were shortlisted and randomized according to the severity of diabetic polyneuropathy, for assessment of the expression of the gene SLC19A3 in stage 1 of the gene expression study. Results. In the first stage of the study in patients with type 2 diabetes with diabetic polyneuropathy with the involvement of small nerve fibers and large nerve fibers we found that the difference in expression of the solute carrier gene SLC19A3 in patients with variable levels of neuropathy was not significant. As evident from the CT values and the value of ΔCT there was no statistically valid difference between the groups. The CT value of the target gene (SLC19A3) in all the three groups, in comparison to each other did not have significant difference in the initial phase of the investigation. In correlation to duration of disease the trend was similar, showing that duration of disease does not play an altering role in the expression of the target gene. In correlation to risk factor glycemic control (level of HbA1c) the expression of the target gene was slightly more profound in patients with an HbA1c of < 8.9 % as compared to the patients with HbA1c value > 9.0 %. Conclusions. Taking into consideration the results of the study it can be stated the expression of SLC19A3 is independent of the severity of diabetic polyneuropathy, duration of diabetes and the glycemic compensation in patients with type 2 diabetes.


INTRODUCTION
Diabetes mellitus (DM) is a chronic progressive disorder which leads to significant disability, morbidity and is likely to progress to become one of the most widespread conditions worldwide and as an additional burden to the healthcare system already reeling under the effects of the COVID-19 pandemic worldwide. According to the International Diabetes Federation Atlas [1] there are approximately 463 million adults (20-79 years) with diabetes mellitus (DM) and it is expected that by 2045 the figure will rise to 700 million. It is noted that the number of patients is increasing in all the countries [2], but around 79 % of the patients live in middle and low income countries.
The pathogenesis of diabetic microvascular complications is complex and marked by metabolic and vascular factors [3,4]. The presence of diabetic polyneuropathy (DPN) in diabetes patients is estimated to be from 6 % to 51 % and depends on various factors such as duration of disease, glycemic control and age [5]. According to published data 50 % of patients can develop a foot ulcer [6,7], and DM is the leading cause of amputation of the limbs [8]. The most common form of DPN is distal symmetric polyneuropathy and consists of large and small fiber neuropathies [9]. It accounts for 75 % of all diabetic neuropathies. During the course of various studies and their results show that DPN is caused by nerve dysfunction due to oxidative stress and inflammatory factors [10].
There is ever increasing evidence regarding the risk factors of DPN in diabetes patients, such as age, duration of disease, obesity, smoking, and presence of chronic kidney disease in patients with type 2 DM and the effect of these risk factors in the progression of DPN.
The treatment of diabetic polyneuropathy is generally supportive and targets the prevention of progression of the disease and its complications. Short term tight glycemic control has not been shown to effectively decrease symptoms of DPN in patients with DM. Results of some studies have shown positive results for treatment of painful peripheral neuropathy [11]. Generally, the recommendations for treatment of DPN are targeted for the relief of symptoms of neuropathy, but current treatment patterns and practices are inconsistent and do not addresses the pathways involved in the pathophysiology of diabetic microvascular complications. Taking into consideration the metabolic pathways involved in the complications of DM, antioxidants are a treatment option [12] ORIGINAL RESEARCH / ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ needs to be further studied. In published data, there is information regarding the effects of benfothiamine, but its effect in the enhancement of the adaptive regulatory response of the expression of THTR-2 have not been elucidated and very little information is available. Therefore, in-depth study of the frequency and the risk factors for the development of DPN in patients with type 2 DM, development of new diagnostic tool for precise, quick and easy assessment of patients with DPN is a promising area, and the use of novel drugs, addressing the pathogenetic mechanisms involved in the progression of DPN is of great practical importance.
The solute carrier (SLC) group of membrane transport proteins include over 400 members organized into 65 families [13]. Most members of the SLC group are located in the cell membrane, but some are located in the mitochondria. The SLC families include genes that encode passive transporters, ion transporters, and exchangers. The different SLC families are functionally related to each other and generally rely on an ion gradient over the cell membrane as the driving force for transportation, but with a few exceptions [14]. The transport protein SLC19A3 (THTR2) is associated with the plasma membrane, mediates the transport of extracellular thiamin into the cytosol. Changes in gene expression by factors such as age has been studied only in animal models [15,16]. In the body the transporter is widely distributed and is consistent with its involvement in thiamin uptake [17]. Very little data is available regarding the expression of thiamine transporter proteins in diabetic patients with DPN, but data regarding the role of THTR-2 and its expression in the intestine and kidneys is available [18].
Objective -to study the correlative variation in gene expression of SLC19A3 in type 2 diabetes patients with proven risk factors of diabetes complications.

MATERIALS AND METHODS
In the study, 190 patients with type 2 DM were screened for diabetic peripheral neuropathy (DPN). DPN was confirmed in (n = 105) patients displaying symptoms of diabetic polyneuropathy with the involvement of small nerve fibers and large nerve fibers. Out of the total cohort, 45 patients with type 2 DM were shortlisted and randomized according to the severity of diabetic polyneuropathy, for assessment of the expression of the gene SLC19A3 in stage 1 of the gene expression study.
The patients were divided into three categories depending upon the severity of DPN. Patients in group 1 (n = 15) had a positive DNS score, positive 5.07-10.00 g monofilament test for testing protective sensory loss and a VPT of 16-25 Vindicating stage 1 DPN. Patients in group 2 (n = 15) had a positive DNS score, positive 5.07-10.00 g monofilament test and a VPT of 26-35 V indicating stage 2 DPN. Patients in group 3 (control) (n = 15) had a positive DNS score but with negative 5.07-10.00 g monofilament test and a normal VPT value of 01-15 V. The patients enlisted for gene expression analysis in different categories of patients did not have any significant difference and statistical variations in the blood tests and the anthropometric data ( Table 1).
All the patients included in the gene expression analysis were of white European ancestry (Caucasian), which in theory corresponds to the Hardy-Weinberg principle of allele and genotype frequencies in a population.
Gene expression analysis of SLC19A3 was examined by RT-qPCR, the material for gene expression testing was the mRNA. The mRNA was extracted from the leucocytes in the peripheral blood of the patients. Extraction and reverse transcription was done with the commercially available SuperScript VILO cDNA Synthesis Kit. It allows to generate the first strand of cDNA for two step RT-qPCR application. The procedure was done according to the procedure recommendations provided by the manufacturer. The next step involved the amplification of DNA in qPCR. In the process TaqMan Fast Advanced Master Mix was used, it contains AmpliTaq Fast DNA Polymerase, uracil-N-glycosylase (UNG), dNTPs with dUTP, ROX dye (passive reference), and optimized buffer components . The primers used for SLC19A3 gene expression were: Forward, 5'-TTCTCC ATGATGAGACCCTC and Reverse, 5'-ATGATGACTGGCTTG TAGCG. The SLC19A3 TaqMan Gene Expression Assay was used to evaluate the gene expression in the samples for the target gene. The expression levels of SLC19A3 mRNA were normalized to glyceraldehyde-3phosphate dehydrogenase (GAPDH). Fold change of

RESULTS AND DISCUSSION
Analysis of SLC19A3 gene expression in patients with type 2 diabetes. In the initial phase (stage 1) of our investigation samples were collected from 45 patients to check the gene expression of the target gene (SLC19A3). The expression levels of SLC19A3 mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ( Table 2).
It is evident that the C T value of the target gene (SLC19A3) in all the three groups in comparison to each other did not have significant difference in the initial phase of the investigation, the difference in expression of SLC19A3 in the groups was ± 2.65 %. In group 1 (C T -26.2667 ± 0.6520) and group 3 (C T -26.6667 ± 0.7080) the C T values for SLC19A3 were quite similar and the expression was more abundant as compared to group 2 (C T -26.7333 ± 0.5380) where the C T value was higher. The scenario for GAPDH was also quite similar to the trend as in the target gene, in group 1 (C T -20.0714 ± 0.6990) and group 3 (C T -19.8667 ± ± 0.8640) the C T value for GAPDH was lower, whereas the C T value in group 2 (C T -20.9333 ± 0.5380) was higher, meaning that the expression was less abundant as compared to the other two groups in the study. The ΔC T μ (mean) for group 1 (with grade 1 VPT loss) was 6,1333 (p < 0.05). The ΔC T μ (mean) for group 2 (with grade 2 VPT loss) was 5.80 (p < 0.05). The ΔC T μ (mean) for group 3 was 6.8 (p < 0.05) (Figure).
The variability of C T values SLC19A3 and GAPDH in correlation to HbA1c in patients with type 2 diabetes in study groups Group 1. The C T values for SLC19A3 in the patients with HbA1c < 7.5 % (25.800 ± 0.656 (± 2.54 %)) and patients with HbA1c 7.6-8-9 % (C T -25.800 ± 1.022 (± 3.96 %)) the C T values were quite similar and the expression was more abundant as compared to patients with HbA1c > 9.0 % (C T -27.200 ± 1.163 (± 4.28 %)) where the C T value was higher. The scenario for GAPDH was also quite similar to the trend as in the target gene patients with HbA1c < 7.5 % (C T -19.600 ± 1.052 (± 5.37 %)) and patients with HbA1c 7.6-8-9 % (C T -19.600 ± 1.052 (± 5.37 %)) the C T value for GAPDH was lower, whereas the C T value in patients with HbA1c > 9.0 % (C T -21.200 ± 0.656 (± 3.09 %)) was higher, meaning that the expression was less abundant as compared to the patients with HbA1c < 8.9 %. The ΔC T μ (mean) for patients with HbA1c < 7.5 % was 6.2. The ΔC T μ (mean) for patients with HbA1c 7.6-8.9 % was 6.2. The ΔC T μ (mean) for patients with HbA1c > 9.0 % was 6.0.
Group 2. The C T values for SLC19A3 in the patients with HbA1c < 7.5 % (26.400 ± 0.894 (± 3.39 %)) and patients with HbA1c 7.6-8-9 % (C T -26.200 ± 0.656 (± 2.50 %)) the C T values were quite similar and the expression was more abundant as compared to patients with HbA1c > 9.0 % (C T -27.600 ± 0.701 (± 2.54 %)) where the C T value was slightly higher. In the case of GAPDH the trend was also quite similar to the target gene in patients with HbA1c < 7.5 % (C T -20.800 ± ± 0.656 (± 3.15 %)) and patients with HbA1c 7.6-8-9 % (CT -20.600 ± 0.701 (± 3.40 %)) the C T value for GAPDH was lower, whereas the C T value in patients with HbA1c > 9.0 % (C T -21.400 ± 1.189 (± 5.56 %)) was higher, meaning that the expression was slightly less profound as compared to the patients with HbA1c for patients with HbA1c < 7.5 % was 5.6. The ΔC T μ (mean) for patients with HbA1c 7.6-8.9 % was 5.6. The ΔC T μ (mean) for patients with HbA1c > 9.0 % was 6.2. Group 3. The C T values for SLC19A3 in the patients with HbA1c < 7.5 % (26.000 ± 1.240 (± 4.77 %)) and patients with HbA1c 7.6-8-9 % (C T -26.400 ± 0.894 (± 3.39 %)) the C T values were quite similar and the expression was more abundant as compared to patients with HbA1c > 9.0 % (C T -27.600 ± 1.052 (± 3.81 %)) where the C T value was slightly higher. This trend was seen in all the groups, that C T value had a tendency to increase in patients with high levels of HbA1c. In the case of GAPDH the trend was also quite similar to the target gene in patients with HbA1c < 7.5 % (C T -19.000 ± 1.839 (± 9.68 %)) and patients with HbA1c 7.6-8-9 % (C T -19.6 ± 0.894 (± 4.56 %)) the C T value for GAPDH was lower, whereas the C T value in patients with HbA1c > 9.0 % (C T -21.000 ± 0.96 (± 4.57 %)) was higher, meaning that the expression was slightly less profound as compared to the patients with HBA1c of < 7.5 % and HbA1c of 7.6-8.9 %. The ΔC T μ (mean) for patients with HbA1c < 7.5 % was 7.0. The ΔC T μ (mean) for patients with HbA1c 7.6-8.9 % was 6.8. The ΔC T μ (mean) for patients with HbA1c > 9.0 % was 6.6.
The variability of C T values SLC19A3 and GAPDH in correlation to duration of disease in patients with type 2 diabetes Group 1. The C T values for SLC19A3 in the patients with duration of disease < 7 years (C T -25.8571 ± ± 0.8330 (± 3.22 %)) was slightly less as compared to patients with duration of disease 7-10 years (C T -26.625 ± 0.913 (± 3.43 %)). The difference in C T values was not significant. For GAPDH there was no significant difference between the two categories. In patients with duration of disease < 7 years (C T -20.1429 ± 1.004 (± 4.98 %)) was almost identical to the C T value of patients with duration of disease 7-10 years (C T -20.125 ± 0.879 (± 4.37 %)). The ΔC T μ (mean) for patients with duration of disease < 7 years was 5.71. The ΔC T μ (mean) for patients duration of disease 7-10 years was 6.5.
Group 2. The C T values for SLC19A3 in the patients with duration of disease < 7 years (C T -26.200 ± 0.656 (± 2.50 %)) and in patients with duration of disease >10 years (C T -26.600 ± 0.894 (± 3.36 %)) was less as compared to patients with duration of disease 7-10 years (C T -27.400 ± 0.894 (± 3.26 %)). The difference in C T values between the categories representing duration of disease did not show any statistical valid trend. For GAPDH in patients with duration of disease < 7 years (C T 20.600 ± 1.052 (± 5.11 %)) and in patients with duration of disease > 10 years (C T -20.800 ± ± 0.656 (± 3.15 %)) was less as compared to the C T value in patients with duration of disease 7-10 years (C T -21.400 ± 0.894 (± 4.18 %)). The difference in C T values in the categories of duration of disease indicated that gene expression of the target gene and the normalizing gene in group 2 was independent of the duration of disease. The ΔC T μ (mean) for patients with duration of disease < 7 years was 5.6. The ΔC T μ (mean) for patients duration of disease 7-10 years was 6.0. The ΔC T μ (mean) for patients duration of disease > 10 years was 5.8.
Group 3 (control). The C T values for SLC19A3 in the patients with duration of disease < 7 years (C T -26.00 ± ± 0.97 (± 3.73 %)) and inpatients with duration of disease >10 years (C T -27.000 ± 1.386 (± 5.13 %)) was less as compared to patients with duration of disease 7-10 years (C T -27.3333 ± 0.9980 (± 3.65 %)). The difference in C T values between the categories representing duration of disease did not show any statistical valid trend. For GAPDH in patients with duration of disease < 7 years (C T -19.1429 ± 1.3390 (± 6.99 %)) the C T value was less as compared to the patients with duration of disease > 10 years (C T -20.500 ± 0.693 (± 3.38 %)) and duration of disease 7-10 (C T -20.5 ± 1.2 (± 5.85 %)). The difference in C T values in the categories of duration of disease indicated that gene expression of the target gene and the normalizing gene in group 3 was independent of the duration of disease. The ΔC T μ (mean) for patients with duration of disease < 7 years was 6.8. The ΔC T μ (mean) for patients duration of disease 7-10 years was 6.8. The ΔC T μ (mean) for patients duration of disease > 10 years was 6.5.

CONCLUSIONS
In the first stage of the study in patients with type 2 diabetes with diabetic polyneuropathy with the involvement of small nerve fibers and large nerve fibers we found that the difference in expression of the solute carrier gene SLC19A3 in patients with variable levels of neuropathy was not significant. As evident from the C T values and the value of ΔC T there was no statistically valid difference between the groups. The C T value of the target gene (SLC19A3) in all the three groups, in comparison to each other did not have significant difference in the initial phase of the investigation, the difference in expression of SLC19A3 in the groups was ± 2.65 %. In correlation to Clinical Endocrinology and Endocrine Surgery / Клінічна ендокринологія та ендокринна хірургія 2 (74) 2021 О ORIGINAL RESEARCH / ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ duration of disease the trend was similar, showing that duration of disease does not play an altering role in the expression of the target gene. In correlation to risk factor glycemic control (level of HbA1c) the expression of the target gene was slightly more profound in patients with an HbA1c of < 8.9 % as compared to the patients with HbA1c value 9.0 %. Taking into consideration the results of the study it can be stated the expression of SLC19A3 is independent of the severity of diabetic polyneuropathy, duration of diabetes and the glycemic compensation in patients with type 2 diabetes. Diabetes mellitus (DM) is a chronic progressive disorder which leads to significant disability, morbidity and is likely to progress to become one of the most widespread conditions worldwide and as an additional burden to the healthcare system already reeling under the effects of the COVID-19 pandemic worldwide.
Aim -to study the correlative variation in gene expression of SLC19A3 in type 2 diabetes patients with proven risk factors of diabetes complications.
Materials and methods. In the study, 190 patients with type 2 DM were screened for diabetic peripheral neuropathy (DPN). DPN was confirmed in (n = 105) patients displaying symptoms of diabetic polyneuropathy with the involvement of small nerve fibers and large nerve fibers. Out of the total cohort, 45 patients with type 2 DM were shortlisted and randomized according to the severity of diabetic polyneuropathy, for assessment of the expression of the gene SLC19A3 in stage 1 of the gene expression study.
Results. In the first stage of the study in patients with type 2 diabetes with diabetic polyneuropathy with the involvement of small nerve fibers and large nerve fibers we found that the difference in expression of the solute carrier gene SLC19A3 in patients with variable levels of neuropathy was not significant. As evident from the C T values and the value of ΔC T there was no statistically valid difference between the groups. The C T value of the target gene (SLC19A3) in all the three groups, in comparison to each other did not have significant difference in the initial phase of the investigation. In correlation to duration of disease the trend was similar, showing that duration of disease does not play an altering role in the expression of the target gene. In correlation to risk factor glycemic control (level of HbA1c) the expression of the target gene was slightly more profound in patients with an HbA1c of < 8.9 % as compared to the patients with HbA1c value > 9.0 %.
Conclusions. Taking into consideration the results of the study it can be stated the expression of SLC19A3 is independent of the severity of diabetic polyneuropathy, duration of diabetes and the glycemic compensation in patients with type 2 diabetes.