【作者】 杜晓晨；

【导师】 罗建明；

【作者基本信息】 广西医科大学 ， 儿科学， 2007， 硕士

【摘要】 目的建立一种简便、可靠的G6PD缺乏症杂合子生化检测方法,并初步探讨其在G6PD缺乏症筛查中的诊断价值。方法在排除地中海贫血、肿瘤、肝病、糖尿病和尿毒症等疾患后,抽取248例非新生儿外周血标本作G6PD酶活性和GR酶活性测定;自然或错配引物介导的聚合酶链反应/限制性内切酶分析130例女性G6PD基因G1376T、G1388A和A95G变异情况。对G6PD显著缺乏的酶切阴性女性分析G6PD基因G871A位点变异情况。用遗传平衡定律计算杂合子的发生率,估计酶切加测序法检出的杂合子在其中所占比例。ROC曲线(receiveroperator characteristic curve,受试者工作特征曲线)法确定G6PD酶直接法和GR/G6PD比值法的最佳工作点(optimal operating point,OOP)。以酶切结合测序的方法为金标准,评价G6PD酶直接法和GR/G6PD比值法对杂合子的诊断价值。各取G6PD酶活性正常、中度缺乏和显著缺乏的三个标本作G6PD酶和GR酶的重复性测定,计算其变异系数。结果本组G6PD酶直接法的参考值范围为8.47～28.19 IU/gHb,2.7～8.46/gHb为杂合子,≤2.6 IU/gHb为显著缺乏。GR酶的正常值为8.44±2.84IU/gHb。109例男性中有14例G6PD缺乏,基因频率为12.84%。用遗传平衡定律进行评估,139例女性中有32例患者(其中30例杂合子);PCR/限制性内切酶分析加测序在130例女性中检出24例杂合子。2例G6PD酶活性显著缺乏的女性未能确定基因型。G6PD酶直接法对杂合子的检出率为26.92%,Youden指数为19.23%,调整一致率为62.34%。经ROC曲线法确定GR/G6PD比值≥0.52为杂合子,此时GR/G6PD比值法对杂合子的检出率为84.62%,Youden指数为49.04%,调整一致率为70.17%,ROC曲线下面积为0.766。经配对卡方比较直接法和比值法对杂合子的诊断价值,结合原始数据认为GR/G6PD比值法对杂合子的诊断价值优于G6PD酶直接法。经ROC曲线法对G6PD酶直接法诊断临界值进行优化,G6PD酶活性≤16.78IU/gHb为杂合子。优化后其对杂合子的检出率为84.62%,Youden指数为40.39%,调整一致率为66.57%,ROC曲线下面积为0.728。经配对卡方比较直接法优化前后对杂合子的诊断价值,结合原始数据认为ROC曲线能够显著改善G6PD酶直接法对杂合子的灵敏度,但不能同时兼顾特异度。GR/G6PD比值法ROC曲线下面积较G6PD直接法ROC曲线下面积大。G6PD酶和GR酶的重复性测定结果的变异系数大多在5%左右;只有G6PD酶活性显著缺乏组的G6PD活性变异系数较大,但仍在显著缺乏范围。结论G6PD酶直接法对杂合子的检出率很低,造成很大一部分的杂合子漏诊。ROC曲线法能显著改善G6PD酶活性直接法对杂合子的灵敏度,但无法兼顾特异度。GR/G6PD比值法对杂合子检测的灵敏度与直接法相同,但特异度高于直接法,其诊断价值高于G6PD酶直接法。基因检测作为参照标准是在杂合子检测评价中第一次采用。更多还原

【Abstract】 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.更多还原