Evaluation Of GR/G6PD Ratio's Accuracy On G6PD Deficient Heterozygote

OBJECTIVE To establish a simple and reliable biochemical method for glucose-6-phosphate dehydrogenase(G6PD)deficient heterozygote and to evaluate its accuracy in the screening of G6PD deficiency.METHODS Two hundred and forty-eight venous blood samples were assayed the activity of both G6PD and GR(glutathione reductase).Thalassaemia, tumor,hepatic disease,diabetes,uremia diseases and neonate samples were ruled out.Three common G6PD deficiency gene point mutation in Chinese(G1376T,G1388A and A95G)was detected in one hundred and thirty females by natural primers or mismatched primers mediated polymerase chain raction follwed by restriction endomuclease analysis(PCR/REA).DNA direct sequencing for G871A genotype was taken to PCR/REA(—)female cases with severe deficiency of G6PD activities.The frequency of heterozygote was calculated by Hardy-Weinberg law,to evaluate the percents of heterozygote which was detected by PCR/REA.The optimal operating point(OOP)of GR/G6PD ratio(GGR)and G6PD activity assay(GAA)were determined by receiver operator characteristic(ROC)curve.PCR/REA combined with direct sequencing was the gold standard for G6PD heterozygote.The diagnostic value on G6PD heterozygote of GP/G6PD ratio and G6PD activity assay was confirmed by the results of PCR/REA.Repeated measurement of both G6PD and GR activity was conducted in our study among three groups:G6PD severe deficiency,G6PD heterozygote and normal.Coefficients of variation(CV)were calculated.Results Normal reference value of G6PD activity in our laboratory: normal was 8.47～28.19IU/gHb,heterozygote was 2.7～8.46IU/gHb,G6PD severe deficient was≤2.6 IU/gHb.While normal reference value of GR activity was 8.44±2.84IU/gHb.Fourteen G6PD deficient cases were detected among 109 male samples,the gene frequency was 12.84%.There were 32 G6PD deficient cases(including 30 heterozygotes)among 130 female calculated by Hardy-weinberg law,and twenty-four heterozygotes were detected by PCR/REA. Two severely deficient female whose genotype were unkown.The actual detection rate of heterozygote by GAA was 26.92%,its Youden's Index(YI)was 19.23%and adjusted agreement(AA)is 62.34%.The OOP of GGR was set to 0.52 by ROC curve,≥0.52 was heterozygote.The actual detection rate of heterozygote by GGR was 84.62%,YI was 49.04%,AA was 70.17%and the area under a receiver operator characteristic curve(AUC)was 0.766.The GGR was superior to GAA on heterozygote detection under the statistical analysis of McNemar's test.The OOP of GAA was set to 16.78IU/gHb by ROC curve,≤16.78IU/gHb was heterozygote.The actual detection rate of heterozygote by GAA rose to 84.62%,YI was 40.39%,AA was 66.57%and the AUC was 0.728.ROC curve couldn't only improve the diagnostic value of GAA to heterozygote significantly but also bring about high false positive rate.The AUC of GGR was large than the AUC of GAA.Most of their coefficient of variation(CV)was about 5%,only the CV of G6PD activity in G6PD deficient group was high but also belonged to severe deficient extent.Conclusion The detection rate of heterozygote by G6PD activity assy was low,thus many heterozygotes were missed.ROC curve could improve G6PD activity assay's sensitivity to heterozygote,but bring about high false positive.The diagnostic value of GR/G6PD ratio to G6PD heterozygote was superior to G6PD activity assay.Genetic analysis was firstly used as the reference standard in G6PD deficient heterozygote detection.