目的 葡萄糖-6-磷酸脱氢酶 （G6PD）缺乏症是最常见的遗传性溶血性红细胞酶缺陷病，绝大多数为编码区单碱基突变，现有的多种基因突变检测方法均存在漏检率。该研究拟探讨RT-PCR测序法检测G6PD缺乏症基因突变的可行性。方法 将2013年8月至2014年7月的195例贫血查因或体检儿童根据G6PD/6GPD比值 （<1.00）分为G6PD缺乏组 （130例）和对照组 （G6PD/6GPD比值≥1.00）65例，优化引物设计及PCR扩增条件，采用RT-PCR测序法对G6PD缺乏组及对照组进行全编码序列分析，基因组DNA测序验证。结果 G6PD缺乏组的基因突变检出率为100%，共检出13种错义突变，包括1种新突变。对照组中28例男性均没有检出错义突变；37例女性中13例检出杂合型错义突变，1例国内外未见报道的纯合型同义突变C1191T，14例C1311T多态位点。对照组的女性标本存在较高的G6PD缺乏症 （携带者）漏检率 （35%，13/37）。结论 RT-PCR测序法对G6PD基因突变检出率高，具有较大临床诊断价值。
Abstract
Objective Since glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary hemolytic erythrocyte enzyme deficiency, most cases have single nucleotide mutations in the coding region, and current test methods for gene mutation have some missed detections, this study aimed to investigate the feasibility of RT-PCR sequencing in the detection of gene mutation in G6PD deficiency.Methods According to the G6PD/6GPD ratio, 195 children with anemia of unknown cause or who underwent physical examination between August 2013 and July 2014 were classified into G6PD-deficiency group with 130 children (G6PD/6GPD ratio <1.00) and control group with 65 children (G6PD/6GPD ratio ≥1.00). The primer design and PCR amplification conditions were optimized, and RT-PCR sequencing was used to analyze the complete coding sequence and verify the genomic DNA sequence in the two groups.Results In the G6PD-deficiency group, the detection rate of gene mutation was 100% and 13 missense mutations were detected, including one new mutation. In the control group, no missense mutation was detected in 28 boys; 13 heterozygous missense mutations, 1 homozygous same-sense mutation (C1191T) which had not been reported in China and abroad, and 14 single nucleotide polymorphisms of C1311T were detected in 37 girls. The control group showed a high rate of missed detection of G6PD deficiency (carriers) in the specimens from girls (35%, 13/37).Conclusions RTPCR sequencing has a high detection rate of G6PD gene mutation and a certain value in clinical diagnosis of G6PD deficiency.
关键词
G6PD 缺乏症 /
基因突变 /
RT-PCR /
基因测序 /
儿童
Key words
Glucose-6-phosphate dehydrogenase deficiency /
Gene mutation /
RT-PCR /
Gene sequencing /
Child
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基金
深圳市科技计划项目（医疗卫生类）重点项目（201201004）。
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