The Establishment And Evalution Of T-ARM-PCR Technology Which Can Detect 6 High Risk G6PD Deficiency Associated Mutations

Objectiveglucose-6-phosphate dehydrogenase deficiency(G6PD) is one of the most common human genetic disease of cellular enzyme. And G6PD gene mutation is chiefly responsible for the G6PD deficiency. Because a number of gene mutation detection procedures are complex,time-consuming, expensive and can not distinguish homozygote and heterozygote,which limiting their use in clinical diagnosis. However Tetra-primer Amplification Refractory Mutation System-Polymerase Chain Reaction is a simple,accurate and specific method,which can also distinguish whether the alleles.This study aims to establish a simple tetra-primer amplification refractory mutation system PCR for detecting 6 point mutations (G1388A> G1376T、A95G、C1004T、C1024T、G871A) associated with G6PD deficiency in China.Methods1.6 positive control plasmids were constructed, which were corresponded with 6 high risk G6PD deficiency associated mutations (G1388A、G1376T、A95G、C1004T、C1024T,G871A).According to design principles of T-ARMS-PCR primers,designed primers of 6 high risk G6PD deficiency associated mutations.2. We established the methodology of tetra-primer amplification refractory mutation system PCR(T-ARMS-PCR) for 6 point mutations of G6PD deficiency with the optimization of primer concentration and annealing temperature.3. We collected samples of G6PD deficiency,then tested DNA samples from patients with G6PD deficiency including 89 patients with G1388A mutation、67 patients with G1376T mutation、37 patients with A95G mutation,245 patients with suspected G6PD deficiency and 50 normal people using T-ARMS-PCR. Finally these samples with mutations were further validated with sequencing.Results1. Positive control plasmids associated with G1388A、G1376T、 A95G、C1004T、C1024T、G871A were successfully constructed and the primers of T-ARMS-PCR were designed.2. The methodology of tetra-primer amplification refractory mutation system PCR(T-ARMS-PCR) for 6 point mutations of G6PD deficiency was successfully established.3. T-ARMS-PCR detected 89 patients with G1388A mutation, including 9 heterozygote and 80 homozygote; 67 patients with G1376T mutation,including 11 heterozygote and 56 homozygote;37 patients with A95G mutation,including 11 heterozygote and 56 homozygote; 13patients with G871A mutation,including 6 heterozygote and 7 homozygote; 16 patients with C1024T mutation,including 5 heterozygote and 9 homozygote.And These results were consistent with the sequencing.ConclusionA user-friendly method for G6PD deficiency associated mutations was successfully developed. It allowed the detection of the six common G6PD deficiency associated mutations with only 6 single tube PCR reactions. In the future,the method could be applied to large population-based epidemiological studies for G6PD deficiency screening.