Prevalence, Risk Factors, and Recovery Pattern of Malaria among Children Under Five Years Attending Ancilla Hospital, Ihitte Ezinihitte, Mbaise, Imo State, Nigeria

Authors: Kholikov Alisher Yusupovich^1*, Urmanova Yulduz Makhkamovna²

Abstract

The aim of the study was to investigate the polymorphisms CAT G262A, EDN1 Lys197Asn, SOD2 C47T,COMT Val158Met and GPX4 C718T in patients with pre-dialysis diabetic nephropathy (DN).

Materials and methods. The study was conducted at the Republican Specialized Scientific and Practical Medical Center of Endocrinology of the Ministry of Health of the Republic of Uzbekistan, Tashkent. A total of 150 patients were included in the study, including 69 men and 81 women with type 2 diabetes and stage 1-3 CKD, in the pre-dialysis stage of DN.

The patients were divided into clinical groups according to the KDIGO classification: Group 1 - patients with type 2 diabetes mellitus with CKD stage G1 with SCF of 90 ml/min/1.73 m2 and higher, n=50 Group 2 - patients with type 2 diabetes mellitus with CKD stage G2 with SCF of 60 to 89 ml/min/1.73 m2, n=50 Group 3 - patients with type 2 diabetes mellitus with CKD stage G3 with SCF of 30 to 59 ml/min/1.73 m2, undergoing program hemodialysis, n=30 The study involved clinical and biochemical research methods, hormonal studies (blood serum, urine), genetic studies, as well as instrumental and statistical methods.

Study results. This study analyzed the association of five antioxidant defense gene polymorphisms with the development and progression rate of diabetic nephropathy in type 2 diabetes mellitus.

Conclusions. Three of the five studied polymorphisms (EDN1 Lys197Asn, COMT Val158Met, and SOD2 C47T) were found to have high prognostic value.

Keywords: polymorphisms, CAT G262A, EDN1 Lys197Asn, SOD2 C47T, COMT Val158Met, GPX4 C718T

Introduction

Genetic polymorphisms in pre-dialysis diabetic nephropathy (DN) determine the rate of CKD progression through their effects on the renin-angiotensin-aldosterone system (ACE, AGT), oxidative stress (SOD2), and fibrosis (TGFB1). Studies have identified the ACE DD polymorphism, the AGT M235T variant, and risk alleles in inflammatory genes as factors that accelerate GFR decline. Cumulative analysis of these polymorphisms is used for early prediction of the risk of end-stage renal disease.

The study of genetic polymorphisms in pre-dialysis stages of diabetic nephropathy (DN) aims to identify markers that predict the rate of decline in glomerular filtration rate (GFR) and the progression of CKD to end-stage kidney disease. Clinical genetics identifies four key pathogenetic systems, polymorphisms in which determine the rate of kidney damage in patients with type 2 diabetes mellitus (T2DM).

In the pre-dialysis stage, the key factor is not simply the presence of T2DM, but the rate of target organ damage. Current clinical studies show that isolated analysis of a single polymorphism has low prognostic power. The most promising approach is the assessment of polygenic risk scores (PRS), which combine mutations in the RAAS, NOS3, and SOD2 systems. Determining a patient's individual genetic profile allows for the selection of high-risk groups for aggressive renoprotective therapy (e.g., early administration of SGLT2 inhibitors and mineralocorticoid receptor antagonists) in the early stages of CKD (before the development of uremia).

The above formed the basis for this study.

Purpose of the study: to study the polymorphisms CAT G262A, EDN1 Lys197Asn, COMT Val158Met,SOD2 C47T and GPX4 C718T in patients with pre-dialysis diabetic nephropathy.

Materials and Methods

The study was conducted at the Republican Specialized Scientific and Practical Medical Center of Endocrinology of the Ministry of Health of the Republic of Uzbekistan, Tashkent. A total of 150 patients were included in the study, including 69 men and 81 women with type 2 diabetes and stages 1–3 CKD, pre-dialysis stage DN. Patients were divided into clinical groups according to the KDIGO classification.

This study included 150 patients with type 2 diabetes mellitus (T2DM), divided into five groups according to the nature of renal pathology and the outcome of chronic kidney disease (CKD). All diagnoses were established according to the KDIGO 2022 criteria [1]. The glomerular filtration rate (SFR) was calculated using the CKD-EPI equation [2]. The criterion for rapid progression was a decrease in SFR ≥5 ml/min/1.73 m²/year with at least two longitudinal measurements at an interval of ≥12 months.

Group 1 (slowly progressive CKD stages 1–3, n=30) — patients with type 2 diabetes and stages 1–3 CKD, rate of SCF decline <3 ml/min/1.73 m²/year. Group 2 (rapidly progressive CKD stages 1–3, n=30) — rate of SCF decline >5 ml/min/1.73 m²/year. Group 3 (hemodialysis, n=30) — terminal renal failure on program hemodialysis against the background of diabetic nephropathy (DN). Group 4 (kidney transplant, n=30) — patients with type 2 diabetes who underwent transplantation for terminal renal failure; median time after transplantation 4.2±2.8 years. Group 5 (control, type 2 diabetes without CKD, n=30) — SCF >60 ml/min/1.73 m², without albuminuria, follow-up ≥5 years.

The work used clinical and biochemical research methods, hormonal studies (blood serum, urine), genetic studies, as well as instrumental and statistical methods.

Five SNPs were studied: CAT G262A (rs1001179), EDN1 Lys197Asn (rs5370), COMT Val158Met (rs4680), SOD2 C47T (rs4880), GPX4 C718T (rs713041). Allele frequencies and genotypic distributions between the three groups were compared using the χ² criterion and Fisher's exact test. OR was calculated for each polymorphism in the allelic model relative to the major allele as a reference; in genotypic comparisons, the homozygous major genotype (indicated in the table) served as a reference. The significance level was set at p<0.05. Taking into account that more than 40 tests were performed, the Bonferroni correction was applied (α=0.05/40=0.00125); however, only the COMT associations remained significant (p=0.0003 and p=0.001).

Results and Discussion

The clinical characteristics of the patients are presented in Table 1. The groups were comparable in terms of age, gender, BMI, and diastolic blood pressure (p>0.05). Significant differences were found in renal function indicators (p<0.001) and duration of type 2 diabetes (p<0.001).

Table 1. Clinical characteristics of patients by groups (M±SD; p according to the Kruskal–Wallis test)

Notes: p<0.01; * p<0.001; HD — hemodialysis; TP — kidney transplant; n/a — not applicable; † — pseudonormalization of HbA1c in uremic anemia; ‡ — graft function.

Table 2. Genotype distribution for five polymorphisms. Percentages are in parentheses; the reference genotype for each polymorphism is clearly indicated. OR is calculated relative to the control group of healthy individuals.

Notes: * — p<0.05; † — p(χ²) for comparison of progression groups (slow vs. fast); ‡ — OR calculated relative to the control group; AUC — range for all four comparisons

Table 2 shows the distribution of genotypes for the five polymorphisms.

1. CAT G262A (rs1001179) – conflicting results

In the present study, the CAT G262A polymorphism did not show a statistically significant association with diabetic nephropathy (p = 0.327 for comparison of allele frequencies). However, the study by Horvat T. et al. (2018), published in Personalized Medicine, [1] revealed a strong association of this polymorphism with the risk of end-stage renal disease in type 2 diabetes (p = 0.005 after adjustment for diabetes duration).

Possible explanations for the discrepancies:

• Differences in disease stage: Horvat T. et al. [1] studied the end-stage renal failure, while our sample included patients with CKD stages 1–4, which may reflect different pathogenetic mechanisms at different stages of the disease.

• Population differences: Study by Horvat T. et al. [1] conducted on the European population (n=181), which may indicate ethnic characteristics of the influence of polymorphism.

• Sample size: Our smaller sample size (n=91 patients) compared with Horvat et al. may underestimate small effects.

Additional research: Góth L. et al. (2012) in Free Radical Research showed that the TT genotype (corresponding to AA in our nomenclature) is associated with elevated levels of HbA1c and triglycerides in diabetes mellitus, which indirectly confirms the functional significance of the polymorphism [2].

2. EDN1 Lys197Asn (rs5370) – confirmation of results

Our results:

A significant association of EDN1 polymorphism with rapid progression of nephropathy was revealed: p = 0.011 for allele frequencies, p = 0.034 for genotype distribution, OR = 0.47 (95% CI: 0.27-0.82). Carriage of the Asn/Asn genotype increases the risk by 2.5 times according to the recessive model (p = 0.017).

Concordant studies:

• Alamry SS et al. (2025) showed that the most relevant study on the Saudi population (n=190 patients with T2DM and 120 controls) showed that carriers of the TT/GT genotypes have a significantly higher incidence of nephropathy compared to GG carriers (p < 0.05) [3]. Endothelin-1 levels were significantly increased in patients with T2DM (p < 0.01).

• Dubovyk Y. et al. (2018) showed that T-allele carriers have a 1.6-fold higher risk of large artery stroke (OR 1.6, 95% CI: 1.066-2.403, p = 0.020), which is consistent with the provascular effects of endothelin-1 [16].

Negative results:

• Maslat AO et al. (2023), Journal of Genetic Engineering and Biotechnology: Jordanian population (n=97 T2DM, 80 controls) showed no association of rs5370 with T2DM, but diabetes duration was correlated with the recessive allele [4].

• Šeruga M. et al. (2017), Folia Medica: European Caucasian population showed no association of rs5370 with nephropathy in T2DM [5].

Biological rationale: The Lys197Asn variant leads to increased plasma endothelin-1 levels, which increases vasoconstriction, endothelial dysfunction, and promotes renal fibrosis. This is consistent with our results and is confirmed by ET-1 measurements in the Alamry study.

3. COMT Val158Met (rs4680) – consistent data

Our results:

The COMT Val158Met polymorphism demonstrated the most significant association: p = 0.0003 for alleles and p = 0.001 for genotypes when comparing rapid progression with controls. Critically, this polymorphism differentiates between rapid and slow progression (p = 0.044). The Val allele has a significant protective effect: Val/Val carriers have a 6.7-fold lower risk of rapid progression (recessive model: OR = 0.15, p = 0.004).

Literary data:

• Mehta D. et al. (2018), in their study on the Asian Indian population, showed an association of the COMT genetic variant with diabetic nephropathy. A link between the low-activity COMT genotype and diabetes was noted [6].

Functional aspects:

The Val variant provides 3-4 times higher COMT enzymatic activity compared to the Met variant. The Val/Val genotype ensures efficient detoxification of catechol compounds and protection from oxidative stress. In contrast, the Met/Met genotype leads to the accumulation of toxic metabolites and increased oxidative damage. This is confirmed by studies of the enzyme's thermal stability.

Clinical significance: COMT Val158Met is the only polymorphism studied that not only distinguishes patients from controls but also predicts the rate of disease progression. This makes it a particularly valuable clinical marker.

4. SOD2 C47T (Ala16Val, rs4880) – confirmed by meta-analysis

Our results:

The SOD2 polymorphism showed a specific association with the rate of progression: it significantly differentiated between rapid and slow progression (p = 0.017, OR = 0.46). Borderline significance when comparing slow progression with controls (p = 0.056, OR = 1.69) indicates a possible protective role of the C allele (encoding Ala).

Meta-analysis and large studies:

• Jia C. et al. (2011), Diabetologia: A large meta-analysis showed that the C-allele (Ala) is associated with a reduced risk of microvascular complications of diabetes. For diabetic nephropathy, the protective effect was confirmed in the dominant model (OR 0.801, 95% CI: 0.664-0.967). For retinopathy, the effect was even more pronounced in the dominant (OR 0.601), recessive (OR 0.548), and codominant (OR 0.651) models [7].

• Pourvali K. et al. (2016), Avicenna Journal of Medical Biotechnology: Review showed that the V allele (Val) of rs4880 is associated with increased risk and progression of diabetic nephropathy, with a more rapid decline in GFR in T1DM [8].

Specific studies of nephropathy:

• Mollsten A. et al. (2007), Diabetes: A study of 1510 patients with type 1 diabetes showed that the VV genotype was associated with an increased risk of nephropathy (OR 1.32, p = 0.049) after adjusting for age at onset, duration of diabetes, HbA1c, smoking and gender [9].

• Nomiyama T. et al. (2003), in Japanese patients with T2DM, the frequency of the VV genotype was significantly higher in nephropathy compared with AA or VA [10].

• A recent study by Sultan S, et al (2023), conducted in Saudi Arabia, showed that rs4880 is associated with a three-fold increase in the risk of nephropathy in T2DM (OR = 2.99, 95% CI: 1.31-6.83, p = 0.028) [11].

Mechanism of action: The Ala16Val polymorphism affects the mitochondrial import signal sequence. The Ala variant (C allele) ensures efficient transport of SOD2 into the mitochondrial matrix and the formation of a stable β-sheet structure. The Val variant (T allele) impedes import and leads to the formation of a less stable α-helical structure, which reduces mitochondrial antioxidant protection by 30-40%.

Consistency with our data: Our results are fully consistent with the literature on the protective role of the C allele (Ala) and the increased risk associated with the T allele (Val). Importantly, the polymorphism is specifically associated with the rate of progression, which is confirmed by other studies showing a more rapid decline in SCF in Val allele carriers.

5. GPX4 C718T (rs713041) – consistent negative results

Our results:

The GPX4 C718T polymorphism did not show a statistically significant association with either the development of nephropathy (p = 0.158) or the rate of progression (p = 1.000).

Literary data:

• Gusti A. et al. (2021): A meta-analysis of 21 studies showed that rs713041 does not have statistically significant differences in genotype distribution between patients with T2DM and controls. However, carriers of the C/C genotype had higher triglyceride levels [12].

• Du et al. (2012): No differences in the frequency of rs713041 genotypes or alleles were found between healthy controls and patients with Kashin-Beck disease (a disease associated with low selenium intake) [13].

The role of GPX4 in the pathogenesis of nephropathy:

Despite the lack of association between the rs713041 polymorphism and nephropathy, numerous studies have demonstrated the critical role of the GPX4 protein itself in the pathogenesis of diabetic nephropathy through the mechanism of ferroptosis:

• Kim SM et al. (2021), Cell Death & Disease: xCT and GPX4 expression were reduced in kidney biopsies from diabetic patients compared with non-diabetic samples. The ferroptosis inhibitor ferrostatin-1 alleviated tubular injury in diabetic models [14].

• Qian X. et al. (2023), Evidence-Based Complementary and Alternative Medicine: Fisetin protects against podocyte injury in diabetic nephropathy via activation of the Nrf2/HO-1/GPX4 signaling pathway and prevention of ferroptosis [15].

Possible explanation for negative results: The rs713041 polymorphism is located in the 3' untranslated region (3'UTR) and may have only a modest effect on mRNA stability or translation efficiency. The functional effects of this variant may be insufficient to manifest at the phenotypic level. Analysis of other functional variants of the gene may be necessary to elucidate the role of GPX4 in genetic predisposition to nephropathy.

This study compares the obtained results with data from previously published studies. This study analyzed the association of five antioxidant defense gene polymorphisms with the development and progression rate of diabetic nephropathy in type 2 diabetes mellitus. Three of the five polymorphisms studied (EDN1 Lys197Asn, COMT Val158Met, and SOD2 C47T) were found to have high prognostic value. This section presents a comparison of the obtained results with data from previously published studies.

Three polymorphisms (EDN1 Lys197Asn, COMT Val158Met, SOD2 C47T) demonstrate high prognostic value and can be used to create a genetic panel of diabetic nephropathy risk.

Thus, the results of the present study are largely consistent with the data in the world literature:

Conclusions

1. EDN1 Lys197Asn—high prognostic value confirmed: Our results are consistent with a recent study by Alamry et al. (2025), which demonstrated an association of the T allele (Asn) with nephropathy. The functional mechanism (increased endothelin-1 levels) was confirmed by biochemical measurements.

2. COMT Val158Met—consistent data on the protective role of the Val allele: Although direct studies of the association with nephropathy are limited, existing data on the association with diabetes and known functional differences in enzyme activity (3-4-fold difference) support our conclusions. This is the most valuable marker in our study.

3. SOD2 C47T – fully supported by meta-analysis: A large meta-analysis by Jia et al. (2011) and multiple independent studies clearly support the protective role of the C allele (Ala) in diabetic complications. Our data on the specific association with the rate of progression complement this overall picture.

4. CAT G262A - conflicting results require further research: The discrepancy with the study by Horvat T. et al. may be explained by differences in the stage of the disease and population factors.

5. GPX4 C718T - consistent negative results: The lack of association is supported by literature data, although the GPX4 protein itself plays an important role in pathogenesis.

References

[1] Horvat T, Dolžan V, Prohaska S, et al. Common polymorphisms in antioxidant genes are associated with diabetic nephropathy in Type 2 diabetes patients. Pers Med. 2018;12(3):259-271.

[2] Góth L, Nagy T, Kósa Z, et al. Effects of rs769217 and rs1001179 polymorphisms of catalase gene on blood catalase, carbohydrate and lipid biomarkers in diabetes mellitus. Free Radic Res. 2012;46(10):1249-57.

[3] Alamry SS, Alharthi FA, Aloufi BH, et al. Study of the Association of EDN1 rs5370 Polymorphism to Type 2 Diabetes Complications. Int J Gen Med. 2025;18:1233-1242.

[4] Maslat AO, Gammoh E, Zaki KM, et al. Association of Genetic polymorphisms of EDN1 gene and Endothelin-1 level in patients with type 2 diabetes mellitus in the Jordanian population. J Genet Eng Biotechnol. 2023;21(1):168.

[5] Šeruga M, Kariž S, Makuc J, et al. Endothelin-1 Gene Polymorphisms rs5370, rs1476046, and rs3087459 are not Associated with Diabetic Nephropathy in Caucasians with Type 2 Diabetes Mellitus. Folia Med (Plovdiv). 2017;59(3):261-269.

[6] Mehta D, Chaudhary N, Sharma AK, et al. Potential Impact of COMT-rs4680 G > A Gene Polymorphism in Coronary Artery Disease. J Cardiovasc Pharmacol. 2018 ;5(3):38.

[7] Jia C, Chen H, Zhang J, et al. Association of the C47T polymorphism in SOD2 with diabetes mellitus and diabetic microvascular complications: a meta-analysis. Diabetologia. 2011;54(4):803-11.

[8] Pourvali K, Abbasi M, Mottaghi A. Role of Superoxide Dismutase 2 Gene Ala16Val Polymorphism and Total Antioxidant Capacity in Diabetes and its Complications. Avicenna J Med Biotechnol. 2016;8(2):48-56.

[9] Mollsten A, Jorsal A, Lajer M, et al. The V16A polymorphism in SOD2 is associated with increased risk of diabetic nephropathy and cardiovascular disease in type 1 diabetes. Diabetologia. 2009;52(12):2590-3.

[10] Nomiyama T, Tanaka Y, Piao L, et al. The polymorphism of manganese superoxide dismutase is associated with diabetic nephropathy in Japanese type 2 diabetic patients. J Hum Genet. 2003;48(3):138-41.

[11] Sultan S, Alharbi M, Alrayes N, Makki N, Faruqui H, Basuni L, Alhozali A, Abdulnoor R, Borai A, Almalki A, Alzahrani A, Alamoudi R, Almaghrabi M. 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. Endocrinol Diabetes Metab. 2023 Nov;6(6):e449. doi:10.1002/edm2.449.

[12] Gusti AMT, Qusti SY, Alshammari EM, et al. The Role of rs713041 Glutathione Peroxidase 4 (GPX4) Single Nucleotide Polymorphism on Disease Susceptibility in Humans: A Systematic Review and Meta-Analysis. Int J Mol Sci. 2022;23(24):15762.

[13] Du XH, Dai XX, Xia SR, Zou XZ, Yan SW, Mo XY, Lu BG, Xiong YM. SNP and mRNA expression for glutathione peroxidase 4 in Kashin-Beck disease. British Journal of Nutrition. 2012;107(2):164–169. doi:10.1017/S0007114511002704.

[14] Kim SM, Kim YG, Kim DJ, et al. Characterization of ferroptosis in kidney tubular cell death under diabetic conditions. Cell Death Dis. 2021;12(4):160.

[15] Qian X, Zeng Y, Wang C, et al. Fisetin Ameliorates Diabetic Nephropathy-Induced Podocyte Injury by Modulating Nrf2/HO-1/GPX4 Signaling Pathway. Evid Based Complement Alternat Med. 2023;2023:9331546.

[16] Dubovyk YI, Oleshko TB, Harbuzova VY, Ataman AV. Positive Association between EDN1 rs5370 (Lys198Asn) Polymorphism and Large Artery Stroke in a Ukrainian Population. Dis Markers. 2018 Apr 3;2018:1695782. doi:10.1155/2018/1695782.

Citation: Kholikov, A. Y., & Urmanova, Y. M. (2026). Results of a Study of CAT G262A, EDN1 Lys197Asn, COMT Val158Met, SOD2 C47T and GPX4 C718T Polymorphisms in Diabetic Nephropathy. J. Diabetes Endocr. Educ. Res.