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

Lawrence  Billy Vasco Djama; Vorthon  Sawaswong; Prangwalai  Chanchaem; Punchalee  Mungkalasut; Thanaporn  Pimpakan; Poonlarp  Cheepsunthorn; Sunchai  Payungporn; Chalisa  Louicharoen Cheepsunthorn

Authors

Lawrence Billy Vasco Djama Medical Sciences Program, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Vorthon Sawaswong Faculty of Science, Mahidol University, Bangkok, Thailand
Prangwalai Chanchaem Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Punchalee Mungkalasut Interdisciplinary Program of Biomedical Sciences, Graduate School, Chulalongkorn University, Bangkok, Thailand
Thanaporn Pimpakan Medical Sciences Program, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Poonlarp Cheepsunthorn Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Sunchai Payungporn Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Chalisa Louicharoen Cheepsunthorn Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Keywords:

3' UTR, G6PD deficiency, glucose 6-phosphate dehydrogenase, miRNA binding, mRNA secondary structure, nanopore MinION sequencing, noncoding mutations, regulatory regions, rs1050757

Abstract

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 within the regulatory regions of the G6PD gene using Nanopore MinION sequencing to investigate the potential effects of noncoding variants on G6PD activity.

Methods: Blood samples from 19 males (13 adults and 6 neonates) with G6PD deficiency or intermediate enzyme activity but with unidentified coding sequence mutations were analyzed. Genomic DNA was amplified using degenerate oligonucleotide-primed PCR and sequenced on the Oxford Nanopore MinION platform. Bioinformatic analyses were performed to evaluate the effects of single-nucleotide polymorphisms on G6PD mRNA structure and miRNA binding potential.

Results: The rs1050757 C > T variant in the 3' untranslated region (3' UTR) of theG6PD gene was detected in 12 individuals, including 9 individuals with intermediate G6PD activity (7 adults and 2 neonates) and 3 neonates with G6PD deficiency. Among adults, G6PD activity was comparable between carriers of the T (11.5 ± 1.3 U/g Hb, n = 7) and C (11.8 ± 0.8 U/g Hb, n = 6) alleles. However, G6PD activity was more variable in neonates, 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 hemolytic anemia were observed in individuals carrying the rs1050757 variant, suggesting a limited direct effect on enzyme function. Computational analyses revealed that the variant induced minor alterations in the secondary structure of the G6PD mRNA, resulting in a shift in the Gibbs free energy (ΔG) from −425.0 to −412.1 kcal/mol and the minimum free energy from−441.1 to −440.4 kcal/mol. These changes may subtly influence mRNA loop formation and miRNA binding at the hsa-miR-92b-3p site.

Conclusion: The rs1050757 variant appears to exert a limited effect on G6PD activity and clinical phenotype. Nevertheless, its potential role in modulating gene regulation through alterations in mRNA structure or miRNA interaction warrants further investigation. Improved understanding of noncoding region mutations may improve diagnostic accuracy and facilitate genotype–phenotype correlations, particularly in malariaendemic regions where G6PD deficiency screening is crucial.

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