Association of a single nucleotide polymorphism in SOD2 with susceptibility for the development of diabetic nephropathy in patients with type 2 diabetes: A Saudi population study

Abstract Introduction One of the complications of diabetes mellitus (DM) is diabetic nephropathy (DN), which plays a significant role in the progression of end‐stage renal disease. Oxidative stress is implicated in DN pathogenesis, and genetic variations in antioxidant enzymes such as superoxide dismutase 2 (SOD2) and catalase (CAT) may contribute to the susceptibility. This study aimed to investigate the potential association between single nucleotide polymorphisms (SNPs) in antioxidant enzymes, specifically SOD2 rs4880 and CAT rs769217, and the risk of T2D and susceptibility to DN within the Saudi population. Methods This case–control study included 150 participants, comprising 50 patients with T2D without DN (group 1), 50 patients with T2D with DN (group 2), and 50 healthy participants (group 3). The samples were genotyped using real‐time PCR for SOD2 rs4880 and CAT rs769217 SNPs. Sanger sequencing was used for validation. Statistical analyses were performed to explore associations between these SNPs and T2D with or without DN. Results No significant difference was observed in CAT rs769217 expression between the groups. However, a significant difference was observed in SOD2 rs4880 expression between the healthy controls and patients with T2D with DN (p = .028). Furthermore, SOD2 rs4880 was associated with approximately threefold increased risk of DN in patients with T2D compared to that in healthy participants (odds ratio [OR] = 2.99 [1.31–6.83]). Validation through Sanger sequencing further confirmed these findings. Conclusions The findings of this study provide evidence that SOD2 rs4880 SNP may contribute to inadequate defence by the antioxidant enzyme, SOD2, against DM‐induced oxidative stress and thus cause DN in Saudi patients with T2D. Therefore, SOD2 rs4880 may serve as a predictive marker to prevent the development and progression of DN in patients with T2D.


| INTRODUC TI ON
Type 2 diabetes (T2D) is a complex disorder associated with high blood glucose levels due to low insulin secretion or inadequate insulin action.In the Kingdom of Saudi Arabia, managing diabetes mellitus (DM) has emerged as a significant challenge, with >50% of the population affected by DM or pre-DM. 1 Between 1992 and 2010, the incidence rate of T2D increased 2.7 times, as reported by the Ministry of Health.Patients with T2D are predisposed to various other chronic diseases and complications, including diabetic nephropathy (DN), neuropathy, retinopathy and cardiovascular disease. 2 DN is a common T2D complication that increases morbidity and mortality rates and plays a significant role in the progression of end-stage renal disease.As previously reported, one in five patients with DM develops end-stage renal disease in the Saudi population. 3nversely, other studies have documented that T2D accounted for 42.5% of end-stage renal disease cases in Saudi Arabia in 2011. 4e progression of DN involves numerous factors that serve as significant markers.These include a reduction in the glomerular filtration rate (GFR) of <60 mL/min/1.73m 2 , proteinuria, poorly controlled blood pressure (>130 mm Hg), high creatinine levels (>130 μmol/L) and retinopathy. 3Recently, diabetic kidney disease had been linked to oxidative stress, which is caused by an imbalance between free radicals and antioxidants.Oxidative stress is accompanied by the overproduction or improper elimination of molecules such as reactive oxygen species (ROS) and reactive nitrogen species.The oxidation-reduction reaction, facilitated by enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase, results in the production of superoxide when an oxygen molecule gains an electron. 5Four pathways implicated in diabetic complications have been identified: protein kinase C, polyol, hexosamine and advanced glycation end products. 6idative stress is involved in inflammation, 7 a significant characteristic of DN. 8 The Care Time study showed increased inflammation in patients with DN. 9 Additionally, various inflammatory markers and cytokines such as mean platelet volume, 10 serum uric acid, 11 monocyte/lymphocyte ratio in hemogram, 12 and neuregulin 13 have been implicated in DN.
Manganese superoxide dismutase (MN-SOD), also known as superoxide dismutase 2 (SOD2), is an important enzyme that eliminates ROS generated during oxidative phosphorylation reactions in the mitochondrial respiratory chain-a significant source of intracellular ROS.Polymorphisms of the SOD2 gene have been identified at amino acid 16 (Val16Ala or rs4880), which may interrupt its ability to eliminates ROS. 14 Catalase (CAT) is a significant enzyme that acts as an antioxidant by degrading hydrogen peroxide to water and oxygen.
Mutations in the CAT enzyme can cause functional defects, leading to neurological disorders such as Alzheimer's disease and Parkinson's disease; metabolic disorders such as DM and hypertension; and cancer. 15Other gene polymorphisms have also been recognized as predisposing risk factors and therapeutic targets for DM with or without kidney diseases.These include sirtuin 1 (SIRT1) rs12778366 and rs3758391, 16 β1-Adrenegic receptor (ADRB-1) rs1801253C/G, 17 NR1H2 gene encoding LXRβ (28,514,894 and rs2303044), 18 miR143/145 cluster, 19 and miR29a (rs157907A/G). 20sufficient cellular antioxidative defence caused by single nucleotide polymorphisms (SNPs) in these antioxidant enzymes may be involved in DN-associated T2D processes.Thus, this study aimed to investigate the potential association between the SOD2 rs4880 and CAT rs769217 SNPs and T2D with or without DN.

| Participants
In this cross-sectional case-control study, blood samples were collected from 150 participants.The participants were divided into three groups: those with T2D but without DN (group 1; n = 50), those with T2D with DN (group 2; n = 50), and healthy participants without diabetes (group 3 [control]; n = 50).These participants sought care at the Diabetes Center in the National Guard Hospital, Jeddah, Saudi Arabia between July 2021 and December 2021.Written informed consent was obtained from all participants after a thorough explanation of the purpose of the study.Additionally, participants were assured that all personal information would be kept confidential.The study was approved by the institutional review board at the National Guard Hospital (IRB#SP21J/419/09).
The control group comprised healthy participants with no history of DM, kidney disease, hypertension, hepatic disease, or other relevant conditions.For group 1, those with a haemoglobin A1c (HbA1c) level ≥6.5%, fasting blood glucose ≥126 mg/dL, 2-h oral glucose tolerance test blood glucose level ≥ 200 mg/dL, and random blood glucose ≥200 mg/dL were classified as having DM, in accordance with the American Diabetes Association criteria from 2011. 21In group 2, participants with an estimated GFR (eGFR) <60 mL/min/1.73m 2 and a urine albumin/creatinine ratio > 30 mg/g were classified as having DM with DN, following the American Diabetes Association criteria. 21The biochemical parameters of the donors were obtained from their medical records.

| Genotype analysis
Whole blood samples (3 mL) were collected from each participant in labelled ethylenediaminetetraacetic acid (EDTA) tubes.Genomic DNA was isolated using the DNeasy kit (Qiagen) as per the manufacturer's instructions and stored at −20°C until further processing.
Genotyping of SOD2 rs4880 and CAT rs769217 antioxidant enzyme polymorphisms was conducted using the TaqMan assay and realtime PCR (Applied Biosystem), following the manufacturer's protocol.Briefly, 20 μL of the mixture was added to each well of a 96-well plate.The mixture included 10 μL of 2× Taq Man Genotyping Master Mix, 1 μL of 20× SNP Genotyping assay, 2 μL of DNA, and 7 μL of H 2 O.The genotyping reference SNP IDs for detection of SOD2 and CAT are rs4880(assay ID:C_8709053_10) and rs769217(assay C_3102907_10), respectively.Subsequently, the microtiter plate was placed in a thermal cycler for enzyme activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and extension at 60°C for 1 min, according to the manufacturer's recommendation.
For validation, Sanger sequencing was used.A mixture of 20 μL was prepared for conventional PCR.The mixture contained 10 μL of the master mix, 1 μL of forward primer and 1 μL of reverse primer.
The forward and reverse primers used for SOD2 rs4880 were GGTAG CAC CAG CAC TAGCAG and TCAGC CTG GAA CCT ACCCTT, respectively.For CAT rs769217, the forward and reverse primers were AAGTA GCG GGA AAG GCAGAA and CACCT GGG GAG CAC CTTTAC, respectively.Subsequently, 6 μL of distilled water (dH 2 O) was added, followed by 2 μL of DNA.A 2% agarose gel was used for electrophoresis, and the labelled gene was detected using a gel imaging system (Azure Biosystem).Exosap (2 μL) was added to 5 μL of the DNA PCR product and incubated at 37°C for 30 min, followed by 80°C for 15 min.The sequencing reaction was performed using 4 μL of Big-Dye, 0.4 μL of primers, 4.6 μL of dH 2 O, and 1 μL of DNA in a total volume of 10 μL.The thermocycler settings were as follows: initial denaturation at 96°C for 1 min (one cycle), followed by 25 cycles of denaturation, annealing, and extension at 96°C for 10 s, 46°C for 5 s, and 60°C for 4 min, respectively.The reaction was then held at 4°C for one cycle.SAM solution (45 μL) (Thermo-Scientific) and 10 μL of beads (Thermo-Scientific) were added to the mixture, followed by vortexing for 45 min at 3000 rpm.The mixture was centrifuged for 2 min at 1000 rpm, and 20 μL of the supernatant was transferred to a 96-well plate, which was sealed and loaded in a Sanger sequencing analyser (Thermo-Scientific).The Snap Gene program was used to analyse the data.

| Statistical analysis
The Statistical Package for Social Sciences (IBM, SPSS, version 28) was used to describe and compare the demographic, clinical, and laboratory findings among the three groups.Qualitative variables were expressed as frequencies and percentages, while numerical continuous variables were expressed as mean ± standard deviation (SD).The Kolmogorov-Smirnov and Shapiro-Wilk tests were applied to explore the normality of quantitative variables.Accordingly, parametric or non-parametric statistical tests of significance were applied.A statistical power of 0.8 was chosen, indicating a 80% probability to correctly reject the null hypothesis.The effect size (d) was calculated by dividing the estimated difference between two compared groups by their pooled estimated SD.The minimum sample size for the study groups was determined using the Raosoft Online sample size calculator. 22This calculation resulted in a minimum requirement of 45 participants per study group, factoring in a 5% margin of error, 95% confidence level, and a reported 3% incidence of DN among patients with T2D. 23However, the sample size was increased to 50 participants in each study group to enhance the sample robustness.χ2 test was used to compare qualitative variables, while Student's t-test and one-way analysis of variance (ANOVA) were used to compare the arithmetic means of continuous variables between two or more groups, respectively.Charts and graphs for diagnostic workup and molecular testing were constructed.Statistical significance was set at p < .05.

| Clinical characteristics of the participants
A total of 150 participants were enrolled in this study, evenly distributed across three groups: T2D without DN (group 1; n = 50), T2D with DN (group 2; n = 50) and control (group 3; n = 50).Their demographic data are summarized in Table 1.

| Comparing the expression of SOD2 rs4880 and CAT rs769217 between healthy participants (control group 3) and those with T2D but without DN (group 1)
As presented in Table 2 and Figures 1 and 2, no statistically significant difference was observed in the expression of SOD2 rs4880 and CAT rs769217 between healthy controls (group 3) and participants with T2D but without DN (group 1).Furthermore, Table 3 presents  The results suggest that the genotype frequencies in each group do not significantly differ from those expected under HWE.This implies that the allele and genotype frequencies for the rs769217 and rs4880 SNPs are likely in equilibrium, and not subject to strong evolutionary forces or selection in this population within two groups (p > .05).
Furthermore, Tables 4 and 5    of participants in the control group expressed AG alleles compared to 67.3% in group 2. Additionally, 26.5% and 32.7% of control participants expressed the AA and GG alleles, respectively, compared to 12.5% and 20.4% of participants in group 2 (p = .028).However, no significant difference was observed between the two groups regarding the expression of CAT rs769217 (Table 6).
The HWE for CAT rs769217 and SOD2 rs4880 SNPs between group 1 and 2 is presented in Table 7.There was no detected deviation from the HWE in either group for CAT rs769217 SNP (p > .05).Additionally, no deviation from the HWE was detected in the control group for SOD2 rs4880 SNP (p > .05).However, a significant deviation from the HWE was observed in group 2 for SOD2 rs4880 SNP (p = .022).

| Comparing the expression of SOD2 rs4880 and CAT rs769217 between participants with T2D but without DN (group 1) and those with DN (group 2)
Table 10 and Figures 1 and 2 present a comparison of the expression of SOD2 rs4880 and CAT rs769217 between groups 1 and 2.
No statistically significant difference was observed in the expression of CAT rs769217 and SOD2 rs4880 between the participants in the two groups (p > .05).No deviation from the HWE was detected in either group for CAT rs769217 (p > .05)(Table 11).However, a significant deviation from the HWE was detected in all participants (p = .013)and in those with T2D and DN (p = .022)for SOD2 rs4880.
Finally, Tables 12 and 13 provide the association analysis of

| Comparing the expression of SOD2 rs4880 with fasting blood glucose (FBG), urine albumin creatinine ratio (UACR), and HbA1c
As shown in Table 14, there was no statistically significant difference when comparing the expression of rs4880 with FBG, UACR and HbA1c.

| Validation by Sanger sequencing
To validate the results, we used Sanger sequencing.Samples from patients with T2D without DN harboured the SOD2 rs4880 A.G heterozygous SNP, and those from the control group were positive for SOD2 rs4880 A.A homozygous SNP, as shown in Figures 3A,B.

| DISCUSS ION
T2D is a metabolic disorder characterized by hyperglycaemia and is associated with several complications, including DN, the leading cause of end-stage renal disease worldwide.Recent studies have revealed an association between SNPs of antioxidant enzymes and improper defence against ROS, leading to intracellular accumulation of free radicals and subsequent oxidative stress. 246][27] Increasing free radicals and improper defence by antioxidants, can cause oxidative damage to lipids, proteins and nucleic acids. 28Genes encoding antioxidant enzymes show varying expression patterns across different populations, with some studies revealing weak or insignificant associations.However, other studies have indicated a significant association in certain populations, along with an increased risk of DN. 24 SOD2 is located in the mitochondrial matrix and functions to convert superoxide free radicals into hydrogen peroxide. 29Polymorphisms involving the substitution of valine with alanine at position 16 in SOD2 have been shown to decrease the formation of active SOD2 in the inner membrane of the mitochondrial matrix of the rat liver. 30other study showed that the alanine variant increased the activity of SOD2 and its ability to neutralize the superoxide free radicals in leukocytes, thereby decreasing oxidized low density lipoprotein and the associated risk of coronary artery disease and myocardial infraction. 31However, under conditions of metabolic disruption such as that in T2D, the increase in SOD2 activity can lead to increased hydrogen peroxide accumulation, contributing to oxidative stress and insulin resistance. 32 this study, we investigated the association between SNPs of the antioxidant enzymes and T2D with or without DN.Our results showed a significant association between SOD2 rs4880 and DN, but not with T2D.Notably, SOD2 rs4880 was linked to a nearly threefold higher risk of DN in patients with DM than in healthy controls (OR = 2.99 [1.31-6.83]).
The results of a previous study conducted on the same population contradicts our results regarding SOD2 rs4880 expression. 33wever, our study included a healthy control group, leading to differences in inclusion criteria.Furthermore, we validated our results using robust Sanger sequencing.Yahya et al. (2019) found that the SOD2 rs4880 polymorphism was associated with development of DN in Malaysian patients with T2D, 36 which aligns with our findings.Another study demonstrated a correlation between SOD2 allelic variants and the onset and progression of DN, indicated by decreased eGFR, elevated plasma advanced oxidation protein products (AOPP) concentration, and reduced SOD2 activity in patients with type 1 diabetes (T1D). 37Collectively, these results indicate that patients with DM with this polymorphism are prone to increased levels of ROS, as reported previously, 35,38 reinforcing the role of oxidative stress in DN among patients with T2D.
In the present study, no association was found between CAT rs769217 and T2D or DN.This finding is similar to that of a previous study that reported no association between CAT rs769217 and acute kidney injury and idiopathic nephrotic syndrome in a Chinese Paediatric Population. 39Similarly, a study involving Caucasian-Brazilian participants found no correlation between the -262C/T polymorphism in the CAT gene and DN. 40However, another study conducted on the Hungarian population to examine the effects of the CAT rs769217 polymorphism on CAT activity found that the activity of the CAT enzyme was significantly decreased in cases of gestational diabetes and T2D. 41dir et al. (2016) also found an association between CAT rs769217 and acute kidney injury. 42CAT rs769217 has been shown to be associated with hospital morbidity and mortality in the Turkish population with acute kidney injury. 42Chistiakov (2006) found an association between the -262 T > C polymorphism of the CAT gene and DN in Russian patients with T1D. 44These discrepancies in findings may be attributed to the ethnic characteristics or exogenous factors contributing to the genotype.
The primary limitation of this study is the small sample size; a larger sample size is needed to confirm our findings.Nevertheless, our study revealed significant differences between healthy controls and patients with T2D with DN regarding the expression of SOD2 rs4880, which encodes the SOD2 antioxidant enzyme.SOD2 rs4880 might increase the genetic susceptibility to DN and may therefore serve as a predictive marker.Our findings may aid in the design of antioxidant therapy to overcome oxidative stress and prevent the development and progression of DN in patients with T2D.
the Hardy-Weinberg equilibrium (HWE) for the CAT rs769217 and SOD2 rs4880 SNPs between participants in groups 1 and 3.The HWE is a crucial concept in population genetics, describing the expected allele and genotype distribution under certain conditions.It assumes factors such as no selection, mutation, gene flow, random mating and large populations to maintain stable allele frequencies.Deviations from HWE may indicate various genetic phenomena such as selection, migration, or genetic drift.

CATF I G U R E 2
rs769217 and SOD2 rs4880 with DN in the study sample.No F I G U R E 1 Distribution of the gene encoding the antioxidant enzyme (SOD2 rs4880) in controls and participants with type 2 diabetes mellitus (T2D) with and without diabetic nephropathy (DN).Distribution of the gene encoding the antioxidant enzyme (CAT rs769217) in controls and participants with type 2 diabetes mellitus (T2D) with and without diabetic nephropathy (DN).
Comparing control subjects and T2D without DN regarding the CAT rs769217 and SOD2 rs4880 SNP.Values represent numbers (percentages), χ2 = Pearson Chi-Square score test, p-Value calculated by a Chi-Square test.
Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; BMI, body mass index; DBP, diastolic blood pressure; EGFR, estimated glomerular filtration rate; FBG, fasting blood glucose; HDL, high density lipoprotein; HbA1c, haemoglobin a1c; LDL, low density lipoprotein; SBP, systolic blood pressure; UACR, urine albumin creatinine ratio.*Statisticallysignificant.TA B L E 1Clinical characteristic of the study subjects.TA B L E 2 Single locus analysis for the association between CAT rs769217 and T2D in co-dominant, dominant, recessive, over-dominant, and log-additive modes (n = 96, crude analysis).Comparing control subjects and T2D with DN regarding the CAT rs769217 and SOD2 rs4880 SNP.Values represent numbers (percentages), χ2 = Pearson Chi-Square score test, and p-value calculated by a Chi-Square test.Values represent numbers (percentages), χ2 = Pearson Chi-Square score test, p-value a calculated by a Chi-Square test, OR = odds ratio, p-value b calculated by simple logistic regression analysis.Comparing the expression of CAT rs769217 and SOD2 rs4880 SNP between group 1 and group 2. Values represent numbers (percentages), χ2 = Pearson Chi-Square score test, and p-value calculated by a Chi-Square test.Single locus analysis for the association between CAT rs769217 and DN in co-dominant, dominant, recessive, over-dominant and log-additive modes (n = 96, crude analysis).
TA B L E 8Single locus analysis for the association between CAT rs769217 and DN in co-dominant, dominant, recessive, over-dominant, and log-additive modes.(n= 98, crude analysis).