B-262 Evaluation of the Genotypic Profile of the c.202 G>A Mutation in the G6PD Gene in Brazil

Abstract

Abstract Introduction Glucose 6 phosphate dehydrogenase (G6PD) is an enzyme found in the cytoplasm of all human cells and plays an important role in preventing cellular damage to reactive oxygen species (ROS). G6PD deficiency is inherited in an X-linked recessive genetic disorder that can destroy red blood cells (hemolysis) leading to acute hemolytic anemia during periods of increased ROS production. The disease prevalence is more frequent in male individuals. To date, more than 400 G6PD variants have been identified, and molecular studies have shown that G6PD deficiency can be associated with different mutations. The c.202 G > A mutation (rs1050828; NM_000402.4) was described as the most frequent. The rapid and accurate diagnosis of this condition is extremely important in neonatal screening and cases of diagnostic suspicion. Therefore, this study aims to describe the genotypic profile of the c.202 G > A mutation in the G6PD gene in Brazilian samples by two molecular assays. Methods Genomic DNA samples from 130 individuals previously genotyped by Restriction Fragment Length Polymorphism PCR were used for validation. DNA isolation was performed in MagNA Pure 24 Total NA Isolation Kit at the automated platform MagNA Pure 24 System (Roche Diagnostics, Basel, Switzerland), according to manufacturer instructions. Genotyping for rs1050828 was performed using TaqMan SNP Genotyping Assays (Thermo Fisher, Foster City, CA) at 7500 Fast Real-Time PCR System (Thermo Fisher, Foster City, CA), according to manufacturer instructions. The results were analyzed using TaqMan Genotyper Software version 1.0.1. Results This study evaluated 130 blood samples, of which 81 were men (62.3%) and 49 were women (37.7%). Male carriers of rs1050828 A genotype (hemizygotes) predominated (56/81, 69.1%) and the remaining carriers for allele G (wild type) were 25/81 (30.9%). While female carriers of rs1050828 GG (wild type) predominated (39/49, 79.6%), heterozygotes were 5/49 (10.2%), and homozygotes also were 5/49 (10.2%). The number of male individuals with the presence of the mutation in hemi/homozygotes was considerably higher when compared to female individuals. This is explained by the fact that the mutation is inherited in an X-linked recessive manner. The results showed agreement and reproducibility of 100% for both methodologies. However, the qPCR assay sensibility and efficiency (>99%) were higher than the RFLP PCR assay. Conclusions Here, we described an efficient molecular method to detect the mutation c.202 G>A in the G6PD gene. The frequency of c.202 G>A described in this study was similar to that observed in others population studies in Brazil. Detection of the mutation target requests an additional evaluation of the G6PD activity level. Obtained results should be evaluated in association with the clinical aspects. The absence of the c.202 G>A in the G6PD gene does not exclude the existence of other mutations in the same region.