Sickle cell anaemia (SCA) is a hereditary haemoglobinopathy characterized by recurrent polymerization of deoxygenated haemoglobin S, erythrocyte deformation, chronic haemolysis, endothelial activation, and progressive vascular injury. Beyond the mechanical consequences of sickling, oxidative stress is now recognized as an important component of disease biology. Autoxidation of haemoglobin S, release of cell-free haemoglobin and haem, catalytic iron, ischemia-reperfusion, leukocyte activation, and reduced nitric oxide bioavailability collectively amplify reactive oxygen species generation and damage erythrocyte membrane lipids and proteins. Iron deficiency anaemia (IDA) is biologically distinct from SCA but can also alter cellular redox homeostasis. Iron depletion is associated with tissue hypoxia, changes in erythrocyte membrane composition, altered activity of antioxidant enzymes, and broader micronutrient disturbances. Consequently, patients with IDA may demonstrate increased lipid peroxidation and reduced antioxidant defence despite the absence of haemolysis-driven oxidative injury. Comparing SCA and IDA within a common analytical framework therefore offers an opportunity to distinguish redox abnormalities associated with haemolytic sickling from those accompanying a predominantly nutritional anaemia. Glutathione peroxidase 1 (GPX1) is a major intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid hydroperoxides using reduced glutathione as an electron donor. GPX1 activity is influenced by selenium availability, oxidative environment, transcriptional regulation, and genetic variation. A common nonsynonymous single-nucleotide polymorphism, rs1050450 (C>T), results in a proline-to-leucine substitution at codon 198 (Pro198Leu). Prior studies in different populations have linked the Leu198 variant with altered GPx activity, although the magnitude and clinical relevance of this association have varied across disease settings. Data on GPX1 Pro198Leu in Indian patients with SCA or IDA are limited, and few studies have simultaneously evaluated genotype, disease status, and measured erythrocyte GPx activity. Establishing a genotype-phenotype relationship is important because it may help explain why antioxidant capacity differs between individuals with apparently similar haematological disease. Such information could also guide future work on redox phenotyping, micronutrient status, and antioxidant interventions. The present study therefore aimed to determine the distribution of GPX1 Pro198Leu (rs1050450) genotypes and alleles in patients with SCA, patients with IDA, and healthy controls; assess the association of the variant with SCA and IDA case status; compare erythrocyte GPx activity across GPX1 genotypes within each diagnostic group; and identify whether genotype independently predicts GPx activity after adjustment for diagnostic group, age, and sex.

Study design and setting: A hospital-based analytical case-control study with three parallel groups was conducted at Siddhartha Medical College, Vijayawada, Andhra Pradesh, India, from December 2023 to November 2025. The genetic and biochemical components were evaluated within the same analytical framework. This project was sponsored by Dr. NTR University of Health Sciences (Dr. NTRUHS), Vijayawada, under its Faculty Research Grant, with Unique Project ID Lr.No:4723/FRG/2023. Ethical considerations: The study protocol was approved by the Institutional Ethics Committee of Siddhartha Medical College and Government General Hospital (IEC approval No. IECSMCGGH/2023/AP/144, dated 20-Nov-2023) before participant enrolment. Written informed consent was obtained from all participants. Coded identifiers were used to maintain confidentiality, and analysis was performed using de-identified data. Study population: Adults aged 18-50 years were screened. The SCA group comprised participants with an HbSS pattern confirmed by haemoglobin analysis who were in steady state at sampling. The IDA group comprised participants with anaemia, a microcytic-hypochromic haematological profile, and biochemical evidence of depleted iron stores, without haemoglobinopathy. Healthy controls had haemoglobin and iron-status measurements within the local reference range and no known haemoglobinopathy. Sample size and participant allocation: The study was planned for a three-group comparison using one-way analysis of variance. Approximately 158 participants were required for a medium effect size (Cohen f=0.25), two-sided alpha of 0.05, and 80% power. The sample was expanded to 180 to support genotype-based and subgroup analyses, resulting in 60 participants in each group. Eligibility criteria: Participants with acute infection or clinically important inflammatory disease, recent blood transfusion or acute sickle-cell crisis, chronic kidney disease, serious chronic liver disease, current malignancy, pregnancy or lactation, ongoing high-dose antioxidant or selenium supplementation, poor-quality samples, unsuccessful DNA extraction, unresolved genotype calls, or incomplete essential clinical information were excluded. Clinical and laboratory assessment: Demographic and clinical data were recorded using a structured case-record form. Complete blood count and red-cell indices were measured using an automated haematology analyser. Reticulocyte percentage was assessed by the laboratory method in use, while haemoglobin fraction analysis was performed when clinically indicated to confirm HbSS or exclude haemoglobinopathy. Serum ferritin was measured by immunoassay, and serum iron and total iron-binding capacity were assessed using routine automated colorimetric methods. Measurement of erythrocyte GPx activity: Erythrocyte GPx activity was measured using a coupled enzymatic assay in which peroxide reduction by GPx consumes reduced glutathione; oxidized glutathione is recycled by glutathione reductase with simultaneous oxidation of NADPH. The decrease in NADPH absorbance at 340 nm was monitored, and enzyme activity was expressed as units per gram of haemoglobin. Assays were performed with calibration and internal quality-control procedures, and samples from all groups were interspersed across analytical runs to limit batch effects. DNA extraction and GPX1 genotyping: Genomic DNA was extracted from EDTA-anticoagulated whole blood using a silica-column method. DNA concentration and purity were assessed spectrophotometrically. A 314-bp fragment encompassing GPX1 rs1050450 was amplified using forward primer 5′-GTGTGCCCCTACGCAGGTA-3′ and reverse primer 5′-CACACAGTTCTGCTGACACC-3′. PCR comprised initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 45 seconds, annealing at 54°C for 45 seconds, extension at 72°C for 45 seconds, and final extension at 72°C for 5 minutes. PCR-RFLP interpretation: The PCR product was digested with ApaI at 37°C and separated on 3% agarose gel. The CC genotype yielded 237-bp and 77-bp fragments; CT yielded 314-bp, 237-bp, and 77-bp fragments; and TT remained as an undigested 314-bp product. A subset of samples was repeated for quality control, and weak or discordant genotype calls were repeated before final coding. Statistical analysis: Continuous variables were summarized as mean ± standard deviation when approximately normally distributed. Categorical variables were compared using chi-square or Fisher exact tests. Allele frequencies were calculated by direct counting, and Hardy-Weinberg equilibrium was evaluated separately in each group. Disease associations were expressed as unadjusted ORs with 95% CIs under dominant and allele-based models. One-way analysis of variance compared GPx activity across genotypes within each diagnostic group. A multivariable linear regression model used GPx activity as the dependent variable and diagnostic group, GPX1 genotype, age, and sex as covariates. A two-sided p value <0.05 was considered statistically significant

A total of 196 individuals were screened; 16 were ineligible or not enrolled, leaving 180 participants for analysis: 60 with SCA, 60 with IDA, and 60 healthy controls. Mean age and sex distribution were comparable across the groups (age p=0.196; sex p=0.501). Table 1. GPX1 rs1050450 genotype and allele distribution Group CC, n (%) CT, n (%) TT, n (%) C allele, n (%) T allele, n (%) HWE p SCA (n=60) 24 (40.0) 28 (46.7) 8 (13.3) 76 (63.3) 44 (36.7) 0.970 IDA (n=60) 31 (51.7) 24 (40.0) 5 (8.3) 86 (71.7) 34 (28.3) 0.907 Control (n=60) 40 (66.7) 18 (30.0) 2 (3.3) 98 (81.7) 22 (18.3) 0.989 The overall genotype distribution differed significantly across the three groups (χ²=9.83, df=4, p=0.043). The TT genotype was progressively more frequent from controls (3.3%) to IDA (8.3%) and SCA (13.3%). The T-allele frequency also increased from 18.3% in controls to 28.3% in IDA and 36.7% in SCA; the overall allele-frequency difference was significant (χ²=10.08, df=2, p=0.006). Genotype frequencies were consistent with Hardy-Weinberg equilibrium in each study group. Table 2. Association of GPX1 rs1050450 with SCA and IDA case status Comparison Genetic model OR 95% CI p value SCA vs control Dominant: CT+TT vs CC 3.00 1.42-6.32 0.006 IDA vs control Dominant: CT+TT vs CC 1.87 0.89-3.91 0.137 SCA vs control T allele vs C allele 2.58 1.43-4.67 0.002 IDA vs control T allele vs C allele 1.76 0.96-3.24 0.093 Carriage of at least one T allele was associated with SCA in the dominant model. Participants with CT or TT genotypes had threefold higher odds of belonging to the SCA group than healthy controls (OR 3.00, 95% CI 1.42-6.32; p=0.006). Likewise, the T allele was associated with SCA on an allele-count basis (OR 2.58, 95% CI 1.43-4.67; p=0.002). Associations for IDA were directionally positive but did not meet conventional statistical significance. Table 3. Erythrocyte GPx activity according to GPX1 rs1050450 genotype Group CC, U/g Hb CT, U/g Hb TT, U/g Hb F p value SCA 32.72 ± 4.99 (n=24) 28.65 ± 5.41 (n=28) 23.58 ± 5.60 (n=8) 9.88 <0.001 IDA 39.51 ± 6.47 (n=31) 36.10 ± 7.38 (n=24) 24.60 ± 2.49 (n=5) 11.07 <0.001 Control 50.92 ± 8.13 (n=40) 46.37 ± 7.96 (n=18) 40.29 ± 4.16 (n=2) 3.27 0.045 Within each diagnostic group, GPx activity decreased in a stepwise pattern from CC to CT to TT. This genotype effect was strongest in SCA and IDA and remained statistically detectable among controls. The consistency of the direction across all three groups supports a genotype-phenotype relationship, although the small number of TT controls warrants cautious interpretation. Table 4. Multivariable linear regression for erythrocyte GPx activity Predictor Adjusted beta, U/g Hb 95% CI p value IDA vs healthy control -11.23 -13.68 to -8.77 <0.001 SCA vs healthy control -17.86 -20.36 to -15.36 <0.001 CT vs CC genotype -3.95 -6.08 to -1.82 <0.001 TT vs CC genotype -11.22 -15.01 to -7.43 <0.001 Male vs female 1.59 -0.40 to 3.57 0.117 Age, per year 0.01 -0.14 to 0.15 0.926 After simultaneous adjustment for diagnostic group, genotype, age, and sex, both anaemia groups retained significantly lower GPx activity than controls. Relative to CC, CT genotype was associated with an adjusted reduction of 3.95 U/g Hb and TT with a reduction of 11.22 U/g Hb (both p<0.001). Age and sex were not significant predictors. The model explained 63.8% of the variability in GPx activity (R²=0.638; adjusted R²=0.626). In the pooled dataset, GPx activity was inversely correlated with malondialdehyde concentration (r=-0.67, p<0.001).

This study identified three convergent findings. First, the GPX1 rs1050450 genotype and allele distributions differed across SCA, IDA, and healthy-control groups, with the T allele most frequent in SCA. Second, T-allele carriage was significantly associated with SCA when compared with controls, whereas the corresponding IDA associations were weaker and statistically non-significant. Third, erythrocyte GPx activity showed a reproducible genotype-dependent gradient, being highest in CC, intermediate in CT, and lowest in TT participants across all three diagnostic categories. The observed genotype-phenotype pattern is biologically plausible. GPX1 is a central peroxide-detoxifying enzyme in erythrocytes, and reduced functional activity could increase susceptibility to lipid and protein oxidation when reactive oxygen species generation is high. Earlier studies have reported associations between the Pro198Leu variant and altered erythrocyte GPx activity in other clinical contexts, although effect estimates have not been uniform across populations. The present data extend this concept to anaemic disorders by demonstrating a consistent decline in measured GPx activity with increasing T-allele dosage. The association between the T allele and SCA should be interpreted as a potential modifier relationship rather than as a determinant of the underlying haemoglobinopathy. SCA is caused by pathogenic variation in HBB; GPX1 does not cause sickle cell disease. A plausible interpretation is that GPX1 variation may modify antioxidant defence after disease is established. This is relevant because the sickle erythrocyte is exposed to continuous oxidant pressure from haemoglobin S autoxidation, haem release, catalytic iron, membrane instability, inflammation, and recurrent ischemia-reperfusion. Under such conditions, genetically lower GPx activity could theoretically intensify lipid peroxidation or reduce resilience to oxidative injury. The IDA group showed an intermediate T-allele frequency and lower GPx activity than controls, but the genetic association with IDA case status did not reach statistical significance. This suggests that the polymorphism may have greater functional relevance in an environment of intense haemolytic oxidative stress than in nutritional iron deficiency, although the study was not powered to prove a difference in genetic effect between the two anaemia types. The comparatively modest association in IDA may also reflect the contribution of nutritional factors, selenium status, hypoxia, and coexisting micronutrient deficiencies to GPx activity. The multivariable analysis strengthened the genotype-phenotype observation. Even after adjustment for diagnostic group, age, and sex, CT and TT genotypes remained independently associated with lower GPx activity. The larger decrement seen with TT than CT is consistent with an allele-dose effect. At the same time, disease category remained a strong predictor, with SCA showing the greatest adjusted reduction. Thus, genetic background and disease-related oxidative burden appear to provide non-redundant information about antioxidant capacity in this cohort. The inverse relationship between GPx activity and malondialdehyde further links the genetic and biochemical findings. Participants with lower GPx tended to have higher lipid-peroxidation levels. However, this pooled correlation is partly influenced by separation between diagnostic groups and cannot establish causality. Stratified analyses, longitudinal sampling, and mechanistic studies are required to determine whether low GPx precedes increased oxidative injury, results from sustained oxidant exposure, or reflects both processes. Several limitations merit emphasis. The study was conducted at a single centre and involved 60 participants per group, limiting precision for uncommon genotypes, particularly the TT genotype among controls. Population stratification and ethnicity were not incorporated into the reported association analysis. Selenium concentration, dietary antioxidant intake, hydroxyurea exposure, transfusion history, inflammatory biomarkers, and detailed haemolysis markers were not included in the multivariable model. The cross-sectional design also prevents causal inference. Finally, PCR-RFLP is suitable for targeted genotyping but sequencing or allelic-discrimination methods would provide additional analytical confirmation. Despite these limitations, the study provides an integrated genotype-phenotype analysis of GPX1 Pro198Leu across two biologically different anaemic states and healthy controls. The consistent reduction in GPx activity across CC, CT, and TT genotypes, together with the higher T-allele frequency in SCA, supports further investigation of GPX1 as a functional modifier of redox status. Larger multicentre studies should evaluate whether rs1050450 is associated with vaso-occlusive crisis frequency, haemolysis severity, organ complications, hydroxyurea response, selenium status, or response to antioxidant interventions.

The GPX1 Pro198Leu (rs1050450) T allele was more frequent among patients with sickle cell anaemia than among healthy controls and was associated with lower erythrocyte glutathione peroxidase activity. GPx activity declined progressively from CC to CT to TT genotypes in SCA, IDA, and controls, and this genotype effect persisted after adjustment for diagnostic group, age, and sex. These findings suggest that GPX1 rs1050450 may function as a genetic modifier of antioxidant capacity, particularly in SCA. Because the study was single-centre and included relatively few TT homozygotes, replication in larger and ethnically characterized populations is required before clinical or predictive use can be considered. RECOMMENDATIONS Future studies should include larger multicentre cohorts, ancestry-informed analyses, direct measurement of selenium and additional micronutrients, and detailed markers of haemolysis and inflammation. Prospective follow-up during steady state and vaso-occlusive episodes may clarify whether GPX1 genotype predicts oxidative burden, clinical severity, treatment response, or benefit from antioxidant-based interventions. Confirmation of PCR-RFLP genotypes by sequencing or allelic-discrimination assays is also advisable

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