琼脂糖凝胶电泳、毛细管电泳及高分辨熔解曲线技术在α-地中海贫血新生儿筛查中的应用 [中文引用][英文引用]

目的 比较毛细管电泳与琼脂糖凝胶电泳2种方法在α-地中海贫血（α-地贫）新生儿筛查中的应用价值；探讨新生儿脐血Hb Bart’s水平与α-地贫基因型的关系；探讨高分辨熔解曲线（high resolution melting，HRM）技术用于筛查非缺失型α-地贫的可行性。方法 对1442例新生儿脐血样本同时采用琼脂糖凝胶电泳及毛细管电泳技术进行血红蛋白定量分析，将其中任意一种方法检出Hb Bart’s区带（其中毛细管电泳技术Hb Bart’s定量最低为0.1%）的样本全部挑出进行Gap-PCR，若样本未检出异常则进一步行HRM分析，随后进行反向点杂交和基因测序。同时对2种电泳Hb Bart’s均为阴性的样本进行2种常见静止型α地贫基因（-α^3.7、-α^4.2）的分子筛查。结果 1442例样本中，2种电泳方法Hb Bart’s均为阳性的样本为151例，基因确诊150例。4例琼脂糖凝胶电泳检测Hb Bart’s阳性而毛细管电泳检测Hb Bart’s阴性的样本，基因检测未见异常。7例琼脂糖凝胶电泳检测Hb Bart’s阴性而毛细管电泳检测Hb Bart’s阳性的样本，基因检测均为静止型α-地贫。1280例两种电泳Hb Bart’s均为阴性的样本检出10例静止型α-地贫。HRM技术将3种常见非缺失型α-地贫全部检出。结论 与琼脂糖凝胶电泳相比，毛细管电泳技术定量分析Hb Bart’s更为简便、准确。脐血Hb Bart’s的含量随着α-基因缺陷的个数的增加而升高，其中α⁺-地贫中不同基因型的Hb Bart’s水平亦存在差异。新生儿脐血毛细管电泳Hb Bart’s含量0.1%应作为区分正常人与α-地贫携带者的指标。HRM技术可作为非缺失型α-地贫（尤其是α^CSα和α^QSα）的一种筛查方法。

Objective  Comparison of automatic capillary electrophoresis(CE) with agarose gel electrophoresis in the value of neonatal screening for α-thalassemia. To investigate the correlation between the levels of Hb Bart’s in cord blood and α-globin genotypes. Explore the feasibility of using HRM to detect the point mutation of non-deletion α-thalassemia. Method  Agarose gel electrophoresis and CE were used to determinate Hb Bart’s amount in cord blood of 1,442 newborns. Samples with the presence of Hb Bart’s were selected for mutant analysis by Gap-PCR. High-resolution melting (HRM) analysis, reverse dot blotting and DNA sequencing was used for samples in which mutations was not detected by Gap-PCR. All of Hb Bart’s negative were selected for genetic testing.  Results  151samples were found positive for Hb Bart’s both agarose gel electrophoresis and CE, and 150 were confirmed by molecular analysis. 4 samples were confirmed normal, with positive for Hb Bart’s by agarose gel electrophoresis but negative for CE. Another 7 samples were confirmed to be α⁺-thalassemia, witch positive for Hb Bart’s by CE but negative for agarose gel electrophoresis. In the 1280 cases of Hb Bart’s negative, 10 samples were confirmed to be α⁺-thalassemia. All of common non-deletional mutations at the exons 3 of the α-globin genes were successfully detected by HRM analysis. Conclusions  To screen for α-thalassemia, CE was proved to be more convenient and effective method as compared to agarose gel electrophoresis. The amount of Hb Bart's was increased with the number of the defective α-genes，and α⁺-thalassemia in different genotypes of Hb Bart’s levels were different  in differet genotype α⁺-thalassemia. In CE testing, 0.1% Hb Bart’s level can be used as a cut-off value for α-thalassemia diagnosis in newborns. HRM is a cheap, rapid and ef?cient screening method for non-deletional α-thalassemia.