Genetic heterogeneity of glucose 6-phosphate dehydrogenase (G6PD) regulatory region in G6PD deficient patients

G6PD deficiency is a genetic disorder caused by either suboptimal function of the G6PD enzyme or instability of the G6PD gene, resulting in insufficient production of the G6PD protein. This study investigates mutations in the regulatory regions of the G6PD gene using Nanopore MinION sequencing, as mutations outside the coding region can also affect G6PD activity. Blood samples from 89 individuals (73 adults and 16 newborns) with G6PD deficiency or intermediate levels but unidentified coding sequence mutations were analyzed. Genomic DNA was screened for mutations, enriched using degenerate oligonucleotide-primed PCR (DOP-PCR), and sequenced using the Oxford MinION platform. Bioinformatic analyzes were conducted to assess the impact of single nucleotide polymorphisms (SNPs) on G6PD gene structure and miRNA binding. The rs1050757 C>T variant in the 3'UTR of the G6PD gene was identified in 76 samples, with 74 exhibiting G6PD intermediate. Females with the T allele had slightly higher enzyme activity compared to those with the C allele (adults: T/T = 11.5±0.8 U/gHb, T/C = 11.6±1.0 U/gHb, C/C = 10.8±0.8 U/gHb; newborns: T/T = 4.9±1.7 U/gHb, T/C = 4.6±2.7 U/gHb, C = 4.3 U/gHb). No cases of hemolytic anemia were observed in patients with the rs1050757 C>T variant. The minor allele frequency (MAF) was 0.233 in adults and 0.231 in newborns, with C being the minor allele. Computational analysis showed that the rs1050757 C>T variant altered the G6PD mRNA secondary structure, changing Gibbs free energy (from ΔG=-421.91 kcal/mol to ΔG=-412.12 kcal/mol) and the minimum free energy (MFE) (from ΔG=-441.1 kcal/mol to ΔG=-440.4 kcal/mol). This mutation affected mRNA loops and the binding site of hsa-miR-92b-3p. The rs1050757 C>T variant in the 3'UTR of the G6PD gene significantly affects mRNA structure and miRNA binding, potentially influencing G6PD activity and highlighting the role of regulatory regions in modulating G6PD expression. These findings underscore the importance of non-coding mutations in understanding G6PD deficiency and its treatment, particularly in malaria-endemic areas.