【摘要】 【目的】探讨中国人肝豆状核变性 (WD)的分子发病机制和基因诊断的方法。【方法】应用基因工程技术对WD进行了 10年的分子生物学研究。【结果】①WD的基因定位研究 :通过RFLP及微卫星多态性分析 ,应用两位点及多位点连锁软件 ,建立了中国人WD基因在D13 q14 .2 -3 区域的精细遗传连锁图谱 ,从而首次对中国人WD基因进行了精确定位 ;②WD基因突变研究 :应用PCR SSCP及DNA测序技术 ,对 39个家系 45名WD患者进行该致病基因的 2 1个外显子突变筛选 ,发现WD基因 5号外显子存在新的T插入突变 ,并证实中国人WD基因的突变热点为 8号外显子 ,突变形式为Arg778Leu ,其频率为 2 2 8% ;③WD的症状前诊断和杂合子检出 :应用DNA重组技术对 79个家系进行基因诊断 ,成功地进行了WD的症状前诊断和杂合子检出 ,并建立了WD的基因筛选的PCR Msp1酶切方法。【结论】中国人WD基因定位与白种人基本相同 ,但基因突变热点不同 ,其发病机制存在差异 ,从而基因诊断的方法也不同。更多还原

【Abstract】 Objective To investigate the molecular basis of hepatolenticular degeneration (Wilson disease, WD) and to attempt to construct the feasibility of gene diagnosis in the disease. Methods We have performed the molecular biological study on this disease for 10 years by molecular genetic techniques. Results ①Location of WD gene in Chinese: Using pairwise linkage analysis and multipoint linkage analysis method, we constructed a genetic map of DNA markers within D 13 q 14.2-3 which refined the location of WD gene by restriction fragment length polymorphism(RFLP) and microsatellite polymorphism analysis; ②Screen for mutations of WD gene in Chinese people: we detected the structure of 21 exons of WD gene in 45 patients from 39 pedigrees by PCR SSCP(Single strand conformation polymorphism) and PCR DNA sequencing technology, found a new mutation in exon 5 and nuclcotide sequence analysis showed it is a T insertion. We also conformed the Arg778Leu in exon 8, the highest frequence mutation point in Chinese people, with mutation rate 22 8% in total;③Carrier detection and presymptomatic diagnosis of WD: Based on DNA recombination technology, we peformed successfully the gene diagnosis in all individuals of 79 families with WD and built up a helpful specific enzyme cut method (PCR Msp1) to detect the carrier and presympomatic patients in Chinese people with WD. Conclusion These results showed that the location of WD gene within D 13 q 14.2-3 is the same in Chinese as in Caucasians, but the gene high mutation point,the gene diagnosis method and its pathogenesis are markly different.更多还原