摘要
目的运用多聚酶链反应-变性梯度凝胶电泳(PCR-DGGE)技术检测葡萄糖-6-磷酸脱氢酶(G-6-PD)缺乏患者及基因携带者基因变异,探讨其对该病的诊断和研究价值。方法提取G-6-PD缺乏症患者及其家系(患者父亲和/或母亲等)的外周血RNA,逆转录合成cDNA后,选取第11至12外显子部分cDNA片段进行PCR-DGGE,观察其电泳行为,将电泳行为异常的标本进行基因测序,最后做出基因诊断。结果36个家系中33个家系发现G-6-PD基因在1304至1520片段出现PCR-DGGE多种异常电泳区带。9例母亲G-6-PD/6-PGD比值低于1.00,其中3例比值低于0.50,而且PCR-DGGE电泳行为一致,基因测序发现为双重杂合子;比值正常的G-6-PD缺乏基因携带者母亲均为单杂合子。该片段基因测序发现3个突变位点分别为:C1311T,G1376T,G1388A。各基因突变的位点有其特殊的电泳行为。结论PCR-DGGE技术是一种敏感性高、可靠性强的筛查基因突变的方法。在临床研究G-6-PD缺乏,特别是常规诊断技术不能发现的女性G-6-PD缺乏基因携带者的检测中具有很强的应用价值。
Objective To detect gene mutations of children with glucose-6-phosphorate dehydrogenase （G-6-PD）deficiency and of carriers of G-6-PD deficiency gene with the technique of polymerase chain reaction and denatured gradient gel electrophoresis （ PCR-DGGE）, and to explore the value of the technique in the diagnosis of G-6-PD deficiency and G- 6-PD deficiency gene carrying. Methods cDNAs were harvested by reverse transcription method after RNAs had been extracted from peripheral blood of 43 children with G-6-PD deficiency and of their family members （ 36 lineages ）. Electrophoresis behaviors of the fragment from exons 11-12 of G-6-PD cDNA were detected with the technique of PCR- DGGE. Gene sequencing was then performed for the abnormal electrophoresis bands. Results Abnormal electrophoresis bands were found in the 1304-1520 fragment of G-6-PD cDNA in 33 out of 36 family lineages. The G-6-PD/6-PGD ratio was below 1.00 in 9 mothers of patients. Three of them had the G-6-PD/6-PGD ratio lower than 0.50. The PCR-DGGE bands were the same in the 3 mothers. Gene sequencing showed double heterozygote in the 3 mothers, but the matemal carriers of G-6-PD deficiency gene who had normal G-6-PD/6-PGD ratio showed mono-heterozygote in gene sequencing. Three mutational sites were found in the 1304-1520 fragment, i. e. , C1311T, G1376T and G1388A. The electrophoresis behaviors were different among the 3 gene mutational sites. Conclusions PCR-DGGE is a sensitive and reliable technique in the screening of gene mutations. It is useful in the diagnosis of G-6-PD deficiency, especially in the diagnosis of female G-6-PD deficiency gene carrying.
出处
《中国当代儿科杂志》
CAS
CSCD
2007年第6期529-532,共4页
Chinese Journal of Contemporary Pediatrics
基金
深圳市科技计划重点项目(200601004)
