Impact of the rs1050757 C>T Variant in the 3'UTR of the G6PD Gene on mRNA Structure and miRNA Binding in G6PD Deficiency: A Nanopore MinION Sequencing Study

Background: Glucose 6-phosphate dehydrogenase (G6PD) deficiency is a genetic disorder caused by impaired enzyme function or instability due to mutations in the G6PD gene, resulting in reduced enzyme activity. This study aimed to investigate mutations in the regulatory regions of the G6PD gene using Nanopore MinION sequencing to explore the potential impact of non-coding variants on G6PD activity. Methods: Blood samples from 19 males (13 adults, 6 neonates) with G6PD deficiency or intermediate enzyme activity but unidentified coding sequence mutations were analysed. Genomic DNA was amplified using degenerate oligonucleotide-primed PCR (DOP-PCR) and sequenced with the Oxford Nanopore MinION platform. Bioinformatic analyses were performed to assess the impact of single nucleotide polymorphisms (SNPs) on G6PD mRNA structure and miRNA binding potential. Results: The rs1050757 C > T variant in the 3' untranslated region (3'UTR) of the G6PD gene was detected in 12 individuals, including 9 individuals with intermediate G6PD activity (7 adults, 2 neonates) and 3 neonates with G6PD deficiency. Among adults, G6PD activity was comparable between carriers of the T allele (11.5 ± 1.3 U/g Hb, n = 7) and the C allele (11.8 ± 0.8 U/g Hb, n = 6). In neonates, G6PD activity was more variable, with mean values of 1.8 ± 4.0 U/g Hb for the T allele (n = 5) and 6.0 U/g Hb for the C allele (n = 1). No cases of haemolytic anaemia were observed among individuals with the rs1050757 variant, suggesting a limited direct impact on enzyme function. Computational analysis revealed that the variant induced minor alterations in G6PD mRNA secondary structure, shifting the Gibbs free energy (ΔG) from -425.0 kcal/mol to -412.1 kcal/mol and the minimum free energy (MFE) from -441.1 kcal/mol to -440.4 kcal/mol. These changes may subtly affect mRNA loop formation and miRNA binding at the hsa-miR-92b-3p site. Conclusion: The rs1050757 variant appears to exert limited influence on G6PD activity and clinical phenotype. However, its potential role in fine-tuning gene regulation through effects on mRNA structure or miRNA interaction warrants further investigation. A better understanding of non-coding region mutations may improve diagnostic accuracy and exploring genotype-phenotype correlations, particularly in malaria-endemic regions where G6PD deficiency screening is critical.