肝豆状核变性患者ATP7B基因8、12号外显子突变的DNA分析

Analysis of the mutation of exon 8 and 12 of Wilson's disease gene

目的对肝豆状核变性(Wilson disease,WD)ATP7B基因的外显子8、12进行DNA测序分析并建立直接基因诊断方法。方法102例WD患者和82例对照正常人提取基因组DNA,PCR扩增ATP7B第8、12号外显子,8号外显子及12号外显子扩增产物分别行MspⅠ及Tail内切酶酶切分析;后对所有患者及对照组DNA外显子扩增产物行直接测序。结果102例WD患者,8号外显子35例存在MspⅠ酶切反应异常,测序示Arg778Leu纯合或杂合突变(占34.31%),其中12例伴Leu770Leu多态性;12号外显子13例存在Tail酶切反应异常,测序示Thr935Met杂合错义突变(占12.75%),1例存在Arg919Gly杂合错义突变,Arg952Lys在患者及对照组中均检出。结论ATP7B基因外显子8、12为WD突变热点,其中8号外显子以Arg778Leu、12号外显子以Thr935Met为主要突变形式,临床上可用PCR-MspⅠ酶切、PCR-Tail酶切反应作为WD患者初步筛选方法。

Objective To sequence and study the exon 8 and 12 of hepatolenticular degeneration（Wilsong disease, WD） gene and to establish the way in direct gene diagnosis. Methods To extract the genomic DNA of 102 WD patients and 82 normal controls, and amplify exon 8 and 12 of ATP7B gene by polymerase chain reaction （PCR）. To analyze the amplification of exon 8 and exon 12 by digestion with Mspl and Tail respectively before sequencing amplification of all the patients and normal controls. Results Digested by MspI through amelioration, 35 cases were abnormal. Sequence results showed that 34.31% of the cases had homozygous or heterozygous Arg778Leu mutation in exon 8, among which 12 were found to have Leu770Leu polymorphism simultaneously; 13 cases were abnormal using digestion by Tail, subsequent direct sequencing showed 12.75% of the cases had heterozygous Thr935Met mutation in exon 12 ,one case was identified as heterozygous Arg19Gly,and Arg952Lys was detected in patients and normal control. Conclusion Arg778Leu in exon 8 and Thr935Met in exon 12 are the hot point mutation of ATPTB gene ,and PCR-Mspl and PCR-Tail digestion are reliable for molecular diagnosis in patients with Wilson＇s disease.