【作者】 孙明然；

【导师】 王峰；

【作者基本信息】 吉林大学 ， 内科学， 2010， 博士

【摘要】 肝豆状核变性又称威尔逊病(WD),是一种因位于13q14.3的ATP7B基因发生位点变异引起的常染色体隐性遗传性疾病。该病全球范围内均有发病,患病率约1:30000~3:100000,针对亚洲黄种人的研究显示,亚洲尤其是中国、韩国及日本的患病率要更高一些。肝豆状核变性病程进展多较快,多于10岁至20岁就可形成不可逆的损伤如肝硬化及神经系统损伤等,严重的危害了人们的健康。该病临床表现常常缺乏特异性,因此早期诊断常是困难的,常导致延误最佳治疗时机。随着分子生物水平技术的发展,直接应用基因测序作为辅助检查手段成为可能,使该病的诊断进入了一个崭新的阶段。1993年成功克隆出ATP7B基因后,学者们针对该病的基因变异进行了大量的研究,现已经发现了近400个致病的基因变异位点及大量的单核苷酸多态性。但由于该病患病率相对低,绝大多都是小样本研究,现仍有很多疑问无法明确解释,如约5%的患者在ATP7B的全部21个外显子内没有或仅有1个致病基因变异位点,拥有相同基因型的患者却可以有完全不同的临床表型等。目前针对我国肝豆状核变性患者的研究多为针对少数几个突变频率高的外显子进行的,极少有对患者整个家系进行系统研究的报道。本研究是通过对肝豆状核变性患者及其亲属的外周血提取基因组DNA,应用相应的引物扩增ATP7B的全部21个外显子,然后对扩增产物直接测序,从而发现位于该基因的变异位点,寻找基因突变热点,为基因诊断提供依据,进而根据患者的临床表现,分析该病的基因型及表现型的关系,并通过对患者家系的基因变异整体分析,寻找可能影响该病因素,为肝豆状核变性的研究及诊断进一步奠定基础。本次研究通过实验,发现5个未见报道的基因变异位点,包括p.V659E,p.S744F和IVS18-1delG 3个考虑为致病的变异位点,IVS1-118ins CGCCG和p.S88S 2个考虑为非致病的变异位点。本次研究结果显示,R778L与肝脏损伤有关,R778L纯合子患者发病年龄相对杂合子早。相对只有2个直接致病的基因突变位点而没有SNPs的患者,具有多个SNPs的患者,其疾病的发生发展相对慢,病情也相对轻一些。而同一个家庭成员具有相同的基因型,临床上却可以有完全不同的表型,提示仍有其他未知的因素影响该病的发生及进展。此外,根据先证者的基因型,对其无法明确是否患病的同胞进行基因检测,是一种快速而准确的诊断方法。更多还原

【Abstract】 Wilson disease is an autosomal recessive disease caused by mutation of ATP7B gene, the mutated gene results in a defect of copper excretion, the overload copper mainly toxic accumulate in the liver, brain and corneas and then results in injures of these organ or tissues. The worldwide prevalence of Wilson disease is estimated to be between 1 in 30,000 and 1 in 100,000, and it is seen more frequently in China. There is a obvious locality difference in ATP7B mutation, almost all the country have its own hot spot mutation. For the white people of European and American, the most common mutation is p.H1069Q which is situated in exon 14, the frequency of this mutation is between 20%- 70%. In China, the hot spot mutation is p.R778L which is situated in exon 8, the frequency of this mutation is between 11.4%-60%. Up to now, most research about chinese Wilson desease patients only study exons which have hot spot mutations, a small part of research do the sequencing of all exons, and also much less research do the whole family members sequencing study. Since the ATP7B gene was cloned by Bulls et al in 1993, a lot of data about the mutational analysis of ATP7B and genotype-phenotype correlation of Wilson disease was published, to date, almost 400 variations reported as causative of WD, but the whole pathologic mechanism of this old diasese is still unclear, as the patients who share the same mutations show the exactly different phenotype, and about 5% Wilson disease patients only have one or non-disease causing mutation.In this work, a total of 17 Wilson disease patients from northeast China are included, 12 of them are male, the other 5 are female. All of them are Han Chinese, four of them are individual patients, the other thirteen patients come from twelve family, and forty family members of those families participate in this study. 4 cc of peripheral blood was drawn from each patient by EDTA tube, and DNA was extracted from these samples by the classic phenol- chloroform method. DNA samples were amplified by PCR using 26 primers corresponding to all 21 exons, and they were subsequently sequenced. After all the exons of the index patients were directly done sequencing, only the exons of patients family members which had found mutations in index patients were done the sequencing. Using direct sequencing methods, 21 different mutations were detected, 10 of them were disease-causing varients, include 9 missense mutation (p.V659E, p.S744F, p.R778L, p.A874V, p.T888P, p.R919G, p.S975Y, p.P992L, p.N1270S) and one splice site mutation (IVS18-1 del G). The other 11 mutations are SNPs, 5 of them are missense mutation (p.A406S, p.L456V, p.K832R, p.R952K, p.A1140V), 2 of them are nonsense mutation (p.S88S, p.L770L), the other 4 mutations are in intron (IVS1-118ins CGCCG, IVS1-74A>C, IVS10-25A>G, IVS18+6 T>C). Thirteen out of 17 patients had two disease-causing mutations; another three had only one mutation found, one patient had no mutation identified. Also, we have identified three new mutations (p.V659E, p.S744F, IVS18-1delG) and two new polymorphisms (IVS1-118ins CGCCG, p.S88S). Using the directly sequencing mehtod, we identified two siblings as Wilson disease patients who were asymptomatic and 28 Wilson desease gene carriers from 12 Chinese Wilson disease pedigrees. Something interesting in this study is that twins with the same genetype and live in the same environment but show up total different phonetype. The prohand patient had both neurological and hepatic symptoms, but his brother has no signs and symptoms. In this cohort, p.R778L was the most common mutation and was identified in 12 WD patients (70.5%), two of them were homozygous of this mutation. In this study, the data suggest that there might be a correlation between p.R778L mutation and liver impairment. An p.R778L homozygote is also at greater risk for early-onset hepatic symptoms. The patients with multiple mutations which suspected non-disease causing, looks like a slowly development pathogenetic condition and lightly level of clinical course compare with patients who only have two disease causing mutations. Wilson disease is an autosomal recessive disease, so, though the direct sequencing of ATP7B gene, we can found the disease causing mutations, then we can screen for prohand patient’s family and find out the carriers and asymptomatic patients. The fact that affected patients within the same family who have the same mutation can have very different phenotypes suggests that there are other factors impacting the presentation of the autosomal recessive disorder.In this study, though the direct sequencing of ATP7B gene, we can found the hot spot Wilson disease causing mutations, and then base on the clinical feature of the patients to find out the genotype-phenotype correlation. And also, though the whole family screen and ATP7B gene analysis, we can try to analyze other factors impacting the presentation of the autosomal recessive disorder to lay foundations for the further work of Wilson disease.更多还原