High resolution melting curve analysis enables rapid and reliable detection of G6PD variants in heterozygous females

Abstract

BackgroundLike glucose-6-phosphate dehydrogenase (G6PD) deficient hemizygous males and homozygous females, heterozygous females could also manifest hemolytic crisis, neonatal hyperbilirubinemia or kernicterus upon exposure to oxidative stress induced by certain foods such as fava beans, drugs or infections. Although hemizygous males and homozygous females are easily detected by conventional G6PD enzyme assay method, the heterozygous state could be missed by the conventional methods as the mosaic population of both normal and deficient RBCs circulates in the blood. Thus the present study aimed to apply high resolution melting (HRM) curve analysis approach to see whether HRM could be used as a supplemental approach to increase the chance of detection of G6PD heterozygosity.ResultsSixty-three clinically suspected females were evaluated for G6PD status using both enzyme assay and HRM analysis. Four out of sixty-three participants came out as G6PD deficient by the enzyme assay method, whereas HRM approach could identify nine participants with G6PD variants, one homozygous and eight heterozygous. Although only three out of eight heterozygous samples had G6PD enzyme deficiency, the HRM-based heterozygous G6PD variants detection for the rest of the samples with normal G6PD enzyme activities could have significance because their newborns might fall victim to serious consequences under certain oxidative stress.ConclusionsIn addition to the G6PD enzyme assay, HRM curve analysis could be useful as a supplemental approach for detection of G6PD heterozygosity.

High-Resolution Melt Curve Analysis: An Approach for Mutation Detection in the TPO Gene of Congenital Hypothyroid Patients in Bangladesh

“…High-resolution melting (HRM) methodology represents a significant advancement in mutation detection over the years. The HRM method has been established for the detection of variants of the beta-globin gene in thalassemia patients and G6PD deficiency in Bangladesh [24, 35].…”

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure

“…With simple design and easy interpretation, our HRM assays can be optimized, and the desired G6PD mutations can be included to enable G6PD genotyping in other areas. In fact, previous studies using different designs and more complicated result analyses demonstrated that HRM assays can detect zygosity in samples ( Yan et al, 2010 ; Islam et al, 2018 ); however, those tests could detect only 1‒2 mutations in a single reaction, making them impractical for use with large sample sizes. The currently available G6PD genotypic platforms appear to have similar limited applications; they are either expensive, time-consuming, or complicated.…”

“…Hence, qualitative methods can correctly identify hemizygous male individuals and homozygous female individuals. However, studies have found that heterozygous female individuals are incorrectly classified as G6PD normal by routine qualitative tests ( Chu et al, 2017 ; Islam et al, 2018 ). Female individuals who are heterozygous for G6PD deficiency exhibit a wide range of G6PD activity levels because they have mixed populations of normal and G6PD-deficient RBCs due to random X-inactivation during embryonic development ( Harper, 2011 ).…”

Cell and Gene Therapy for Anemia: Hematopoietic Stem Cells and Gene Editing

“…G6PD deficiency diagnosis can be made in women homozygous for G6PD gene mutations [89,95]. Moreover, women heterozygous for G6PD gene mutations may give birth to hemizygous male infants who could develop hemolytic crisis upon inadvertent exposure to oxidative stress, which could induce kernicterus, hyperbilirubinemia, and even death [96].…”