CAJ | 학술논문

目的对多色探针荧光PCR熔解曲线法用于葡萄糖-6-磷酸脱氢酶缺乏症G6PD基因突变检测进行临床评价.方法收集402份疑似患者或其家系成员的外周血样本(男256例、女146例),先用酶学方法(G6PD/6PGD定量比值法)进行G6PD缺乏症初筛,经基因DNA提取后,按双盲对照试验,分别应用多色探针荧光PCR熔解曲线法(可检测16种G6PD基因突变)和DNA测序法对各样本进行G6PD基因突变检测,比较两种方法基因突变的符合率.结果酶学检测发现G6PD/6PGD比值＜1.0的样本有170例,G6PD/6PGD比值≥1.0的样本232例.应用DNA测序法检出182例野生型样本,151例半合子突变型样本,5例女性纯合突变型样本,54例女性杂合突变型样本和10例女性复合杂合突变型样本;使用多色探针荧光PCR熔解曲线法检出185例野生型样本,148例半合子突变型样本,5例女性纯合突变型样本,55例女性杂合突变型样本和9例女性复合杂合突变型样本.多色探针荧光PCR熔解曲线法检测G6PD基因突变的特异性为100％(182/182),灵敏度为98.6％(217/220),阳性预测值为99.5％(216/217),阴性预测值为98.4％(182/185),约登指数为0.986,总符合率为99.0％(398/402).多色探针荧光PCR熔解曲线法共检出21种基因型,DNA测序法检出24种基因型.有4例样本与DNA测序法的基因型检测结果不相符,主要原因为这些样本的G6PD基因突变不在多色探针荧光PCR熔解曲线法所设计的检测范围内.结论荧光PCR熔解曲线用于G6PD基因突变的检测,具有简便、快速、灵敏度高、特异性强等优点,可用于G6PD缺乏症的临床辅助诊断.
목적 대다색탐침형광PCR용해곡선법용우포도당-6-린산탈경매결핍증G6PD기인돌변검측진행림상평개.방법 수집402빈의사환자혹기가계성원적외주혈양본(남256례、녀146례),선용매학방법(G6PD/6PGD정량비치법)진행G6PD결핍증초사,경기인DNA제취후,안쌍맹대조시험,분별응용다색탐침형광PCR용해곡선법(가검측16충G6PD기인돌변)화DNA측서법대각양본진행G6PD기인돌변검측,비교량충방법기인돌변적부합솔.결과 매학검측발현G6PD/6PGD비치＜1.0적양본유170례,G6PD/6PGD비치≥1.0적양본232례.응용DNA측서법검출182례야생형양본,151례반합자돌변형양본,5례녀성순합돌변형양본,54례녀성잡합돌변형양본화10례녀성복합잡합돌변형양본;사용다색탐침형광PCR용해곡선법검출185례야생형양본,148례반합자돌변형양본,5례녀성순합돌변형양본,55례녀성잡합돌변형양본화9례녀성복합잡합돌변형양본.다색탐침형광PCR용해곡선법검측G6PD기인돌변적특이성위100％(182/182),령민도위98.6％(217/220),양성예측치위99.5％(216/217),음성예측치위98.4％(182/185),약등지수위0.986,총부합솔위99.0％(398/402).다색탐침형광PCR용해곡선법공검출21충기인형,DNA측서법검출24충기인형.유4례양본여DNA측서법적기인형검측결과불상부,주요원인위저사양본적G6PD기인돌변불재다색탐침형광PCR용해곡선법소설계적검측범위내.결론 형광PCR용해곡선용우G6PD기인돌변적검측,구유간편、쾌속、령민도고、특이성강등우점,가용우G6PD결핍증적림상보조진단.
Objective To evaluate the clinical value of multicolor melting curve analysis(MMCA) for detecting genetic mutations in G6PD deficiency.Methods A total of 402 peripheral blood samples(256 males and 146 females) were collected from suspected patients or their relatives at the Prenatal Diagnosis Center of Liuzhou Maternal and Child Health Hospital between March 2012 and May 2012.The samples were screened by G6PD/6PGD quantitative ratio testing.The reliability of the assay was evaluated by multiplex probe melting curve assay(which can detect 16 G6PD mutations) and DNA sequencing through a double blind study.Results One hundred seventy cases with G6PD/6PGD ratio＜1.0 and 232 cases with G6PD/6PGD ratio≥1.0 were detected by the enzymological method.DNA sequencing has identified 182 wild type samples,151 hemizygous mutation samples,5 female homozygous mutation samples,54 female heterozygous mutation samples and 10 female double heterozygous mutation samples.Multicolor melting curve analysis has detected 185 wild type samples,148 hemizygous mutation samples,5 female homozygous mutation samples,55 female heterozygous mutation samples and 9 female double heterozygous mutation samples.The specificity and sensitivity of G6PD gene mutation detection by multicolor melting curve analysis were 100％ (182/182) and 98.6％ (217/220),respectively.The positive predictive value and negative predictive value were 99.5％ (216/217) and 98.4％ (182/185),respectively,and the Youden's index was 0.986.The concordance rate of the sample detection between the melting curve assay and DNA sequencing was 99.0％ (398/402).Twenty-one different genotypes were detected by the multicolor melting curve analysis and 24 different genotypes were detected by DNA sequencing.Four samples containing mutations(c.196T＞A or c.406C＞T) were not detected by multicolor melting curve analysis,which can be attributed to different technical settings of the two methods.Conclusion Multicolor melting curve analysis for G6PD gene mutation detection is a simple,rapid,sensitive and specific method,which can be used for clinical diagnosis of G6PD deficiency.