摘要
目的 了解广东人葡萄糖 6 磷酸脱氢酶 (G 6 PD)缺陷的基因突变型。探讨干血纸片法用于基因突变型诊断的效果。方法 采用专用滤纸干血斑 ,运用 5对特异性引物进行DNA直接扩增、结合限制性内切酶分析技术对 87例G 6 PD缺陷的住院患儿进行检测。结果 87例患儿中 30例(35 % )为G1376T ,2 8例 (32 % )为G1388A ,6例 (7% )为A95G ,1例 (1% )为G392T ,未检出C10 2 4T ,尚有2 2例未能定型。结论 G1376T、G1388A是广东人最常见的G 6 PD突变型。对急性溶血患儿 ,高铁血红蛋白还原率和G 6 PD酶活性 (NBT法 )假阴性时 ,干血纸片法可提高检出率。滤纸干血斑标本采集容易 ,直接进行DNA扩增 ,方法简单 ,准确度高 ,值得在基因研究和临床诊断中应用推广。
Objective To understand gene mutant types of glucose 6 phosphate dehydrogenase (G 6 PD) deficiency among local Guangdong children and to evaluate the method using dried blood spots on filter paper in examination of patients suffered from acute hemolytic anemia caused by G 6 PD deficiency. Methods The subjects of this study were 87 hospitalized children suffering from acute hemolytic anemia episodes between 1993 and 1997. Among these children, 76 were male and 11 were female. The age of these cases ranged from 1.5 mo to 10 years and 7 mo. All the patients were diagnosed as having G 6 PD deficient disease on the basis of clinical symptoms and the methemoglobin reducing test or the activity of G 6 PD enzymes (NBT method). All these patients were native Guangdong people without blood relation to each other. Peripheral blood specimens were collected from the finger tips of the patients, then the blood was dropped on a sheet of special filter paper (S&S) and the detection was performed after dry blood spot formed. Direct PCR amplification of the target gene segment was performed by using 5 pairs of special primers designed against 5 G 6 PD mutant types: G1376T, G1388A, A95G, G392T, and C1024T from dried blood spot on filter paper. The amplified products were added with related 5 different restriction endonucleases: BfrⅠ, NdeⅠ, MIuⅠ, BanⅠ, MboⅡ, and were digested at 37℃ overnight. The products obtained after digestion were analyzed with polyacrylamide gel electrophoresis. Results The detection rates of different gene mutant types were as follows: G1376T 35% (30 cases), G1388A 32% (28 cases), A95G 7% (6 cases), G392T 1% (1 case); C1024T was not found. In 22 cases the mutant types could not be defined. Of 7 cases with acute hemolytic anemia who had normal G 6 PD enzyme activity (NBT method), 5 were G1376T positive, one was G1388A positive and the remaining one was A95G positive. Conclusion G1376T and G1388A seemed to be the most common mutant types of G 6 PD among the subjects studied. The method of using dried blood spots on filter paper was useful for the patients suffered from acute hemolytic anemia caused by G 6 PD deficient disease. This method may have higher detection rates than methemoglobin reducing test and the G 6 PD enzyme activity tests which often show false negative results. The dry blood spot test has the advantages of easy to perform, using small quantity of blood sample, convenient to transport and store, which would make direct DNA amplification simple and accurate. This method appears to be suitable for mass screening for G 6 PD deficiency.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2000年第7期421-423,共3页
Chinese Journal of Pediatrics
关键词
基因突变
干血纸片法
G6PD缺乏
Glucosephosphate dehydrogenase deficiency
Genotype
Mutation
Polymerase chain reaction
DNA restriction enzymes
