Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh.

Fig 1. Proportion of G6PD enzyme activity levels for male and female participants compared to adjusted male median value.

For each G6PD enzyme activity level (U/g Hb) shown in X-axis, the corresponding value in Y-axis indicates the number of participants. The numbers on top of each dotted line, shown as 10, 20, 30, and 60 on uppermost horizontal line of the graph indicate different cut-off values as percentages for the study population. 60% (shown as 60) of adjusted male median (shown as 100) is the upper limit of cut-off value and the participants with enzyme activities below 60% are considered deficient. Black portion of each bar indicates male participants, whereas gray portion of each bar indicates female participants.

Fig 2. Hemoglobin levels and reticulocyte counts in G6PD non-deficient (ND) and deficient (D) participants.

(A) Depicts hemoglobin levels (g/dL), and (B) Demonstrates reticulocyte counts (%) in non-deficient and deficient participants. A p-value < 0.05 was considered statistically significant.