74例肝豆状核变性患者中ATP7B基因七种新突变的发现

7 novel mutations detected in 74 patients with Wilson's disease

目的分析中国人肝豆状核变性(Wilsondisease,WD)患者ATP7B基因突变的分布特征,建立利用变性高效液相色谱(DHPLC)技术对Wilson病进行基因诊断的方法,并评价其在临床的应用价值。方法对临床确诊为Wilson病的74例患者及50名健康人抽取外周静脉血提取基因组DNA。以患者和健康人的DNA为模板,分别对ATP7B基因的21个外显子进行PCR扩增。取PCR产物应用DHPLC技术在部分变性条件下检测突变并DNA测序证实突变位点。结果利用DHPLC技术筛查并经测序证实,共发现22种ATP7B基因突变类型,其中7种是新发现的,同时发现11种多态,其中3种是新发现的。第8外显子Arg778Leu突变率最高,其频率为25.0%。其次,2356-2A>G突变频率为3.4%,Arg919Gly突变频率为2.7%,其他外显子突变频率均在1.0%～2.0%之间。结论中国人的WD基因突变具有多样性特点,热点突变是第8外显子Arg778Leu。DHPLC具有高通量、敏感、准确且经济的特点,适合于大样本的筛查且能够发现未知的突变,是一种有临床应用价值的基因诊断技术。

Objective To investigate the characteristics of distribution of mutations of WD （ATI：rTB） gene in Chinese patients with Wilson＇ s disease, and to optimize the technique of denaturing high- performance liquid chromatography （DHPLC） for detecting mutations of WD gene. Methods Genomic DNA of 74 Chinese patients with Wilson＇ s disease and 50 normal controls were subjected to polymerase chain reaction （PCR） and mutations of ATPTB gene coding regions were detected by DHPLC and sequencing. Results Twenty-two mutations were found, including seven novel mutations. Eleven polymorphisms, including three novel ones, were found . All above novel mutations and polymorphisms were deposited in GenBank with the Accession Numbers AY972085-972097. The mutation frequencies for Arg778Leu, 2356- 2A 〉 G and Argg19Gly were 25.0% , 3.4% and 2. 7% , respectively, and the frequencies of other mutations only accounted for 1.0% to 2. 0%. Conclusion There is a wide spectrum of mutation profile and the exon 8 Arg778Leu mutation is the hot spot for Chinese patients with Wilson＇ s disease. DHPLC is a dependable tool for ATP7B screening that is superior to traditional method and allows fast, sensitive, and specific detection for known and unknown mutations.