摘要
目的探讨高分辨率熔解曲线（high-resolutionmelting，HRM）分析技术用于citrin缺陷导致的新生儿肝内胆汁淤积症（neonatalintrahepaticcholestasiscausedbycitrindeficiency，NICCD）筛查和诊断的可行性。方法根据中国人群SL（225A13基因的热点突变类型（851del4、1638ins23、IVS6＋5G〉A和IVS16ins3kb）设计特异性HRM扩增引物，挑选经测序证实的50名正常对照和20例NICCD患儿，建立和完善HRM检测条件。用优化后的HRM检测方法对171例临床疑似的NICCD患者进行HRM筛查。若受检样本的熔解曲线与阳性质控样品相吻合，则进行DNA测序分析。结果优化后的HRM方法能准确地对50名正常对照及20例NICCD患儿进行基因分型，其灵敏度和特异性均为100％（70／70）。重复实验表明，相同基因型的不同样品HRM熔解曲线完全吻合，重复性好。在171例疑似患儿中，有7例患儿熔解曲线与阳性质控样品基因型相符，其HRM基因分型为：1例851del4纯合突变，1例IVS6＋5G〉A杂合突变，3例851del4杂合突变，1例[IVS6＋5G〉A]＋[851del4]，1例[1638ins23＋IVS16ins3kb]＋[1638ins23]。DNA测序证实了HRM基因分型，准确率为100%。结论HRM技术具有高通量、操作简便、结果准确、重复性好等优点，可对临床疑似的NICCD患儿进行基因筛查和诊断。
Objective To assess the feasibility of high-resolution melting （HRM） analysis for screening patients with neonatal intrahepatic eho[estasis caused by citrin deficiency （NICCD）. Method Based on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations （851del4, 1638ins23, IVS6＋5G〉A and IVS16ins3kb） were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing. Results HRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% （70/70）. The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations included one case of 851de14 homozygote, one case of IVS6＋5G;A heterozygote, 3 cases of 851de14 heterozygotes, one case of [IVS6＋5G〉A]＋[851de14] and one case of [1638ins23＋IVS16ins3kb]＋[1638ins23]. All mutations were subsequently confirmed by DNA sequencing. Conclusion HRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2012年第2期167-171,共5页
Chinese Journal of Medical Genetics
基金
基金项目：深圳市医学重点学科专项经费（2001B18、2001B19）
