Screening of G6PD Mutation and polymorphism using high resolution melt curve analysis

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common X-linked recessive disorders resulting from the defect of a single gene encoding an enzyme called G6PD. G6PD enzymes catalyzes the conversion of glucose-6 phosphate into 6phosphoglucono-δ-lactone, which is a rate limiting step in pentose phosphate pathway (PPP).PPP supplies reducing energy to red blood cells (RBCs) by maintaining NADPH levels which in turn maintains reduced glutathione levels. Reduced glutathione protects RBCs against oxidative damage. This disorder can cause potentially life threatening hemolytic anemia under conditions of oxidative stress due to consumption of certain foods, drugs, or infections. In 2013, 4100 deaths occurred globally due to G6PD deficiency. The prevalence rate and the cut off values of G6PD in Bangladesh are not yet established. Our unpublished data generated through G6PD gene sequencing of G6PD deficient patients demonstrate that three different mutations, namely G6PD Mahidol (c.G487), Kalyan-Kerala (c.G949) and Orissa (c.C131G) are prevalent in Bangladesh. However, gene sequencing is a time consuming and costly procedure to identify the genetic basis of G6PD deficiency. To overcome the difficulties, a new method using real time PCR-based HRM analysis could successfully identify all mutant alleles based on different melting curve shapes. Six G6PD deficient samples were subjected to HRM analysis using four pairs of G6PD gene-specific primers. Among six mutant alleles, five were found to have a c.G487A (Mahidol) mutation and one had the c.G949A (Kalyan-Kerala) mutation and the results were consistent with sequencing data, confirming the validity of the procedure. The sensitivity and specificity of mutation detection was 100% for c.G487A and c.G949Amutations. For other six G6PD deficient specimens that had c.C131G mutation, PCR-HRM approach could not be applied due to lack of specific primers for that region. However, it is expected that the primer – specific real time PCR-HRM approach would definitely work for c.C131G mutation. The results also demonstrate the usefulness of real time PCR-HRM analysis for detection of heterozygous alleles for c.1311T/C and IVS-11_93t/c polymorphisms of G6PD gene. The findings elucidate a promising potential for use of G6PD primer-specific real time PCR-HRM analysis as a faster and cost effective approach to identify known G6PD mutations and polymorphisms that are prevalent in Bangladeshi population.