WD基因突变热区和高频突变位点及特殊类型WD基因诊断的研究
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摘要
课题背景
肝豆状核变性(Hepatolenticular degeneration, HLD)又名Wilson病(Wilson's disease, WD),是一种常染色体隐性遗传性铜代谢障碍疾病,不同人群的发病率约为15～30/100万,其基因定位于13q14.3, cDNA编码由1465个氨基酸组成的分子量约165Kda的一种P型铜转运ATP酶(copper-transporting P-type ATPase, ATP7B)。WD患者由于ATP7B基因突变使位于肝细胞高尔基反面网状结构(trans-Golgi network, TGN)及胆管膜侧的ATP7B酶功能缺陷,引起铜离子跨膜转运障碍,导致铜蓝蛋白(ceruloplasmin, CP)合成障碍及胆汁中铜的排泄受限,过量的铜不能排泄出体外而在肝、脑、肾、角膜等部位沉积并引起相应的组织脏器损伤,患者可出现血清CP水平减低、肝硬化、神经/精神症状、角膜K-F环等临床表现。
WD是少数可以治疗的神经遗传病之一,患者如果能在发病早期或症状前期被确诊并得到及时治疗,大多预后良好,反之病情逐渐加重甚至危及生命。但由于铜在不同脏器中沉积的速度和程度不同,许多患者早期症状复杂多样,极易被误诊为其他疾病,实验室铜代谢检查等又存在假阳性或假阴性结果,因此本病的早期诊断特别是症状前期和产前诊断较为困难。WD基因的克隆,使本病患者通过基因诊断技术进行症状前期及产前诊断成为可能。
大量研究结果证实,WD基因存在高度的遗传异质性,各外显子的突变位点众多,目前已发现超过300种形式的基因突变,且多为复合杂合突变,常见突变形式少见,多为罕见突变,在不同人种的突变特征亦存在明显差异。这虽然解释了WD患者临床表现复杂多样的原因,但给基因诊断走向临床实用带来了困难。因此,明确不同地域、不同人群WD基因的突变热区和高频突变位点并采用PCR或多重PCR技术扩增相应人群的突变热区并进行序列比对分析,是检出WD患者的一种快速、高效的基因诊断策略。
由于WD患者肝脏中铜的沉积通常经历肝铜蓄积期、肝铜饱和释放期等病理过程,大多在5～35岁之间起病。但近来的多个流行病学调查研究发现,40岁以上起病的晚发性WD患者并不少见。血清CP水平减低是WD的特征性生化改变,也是最主要的诊断依据,但约有5%左右的WD患者血清CP水平在正常范围。由于这两种特殊类型的WD患者在临床中极易被误诊误治,常给临床医生的WD诊疗工作带来困难,而基因诊断可为其提供最有力的诊断依据。
血清CP由与铜离子结合的全CP(Holo-ceruloplasmin, Holo-CP)和未与铜离子结合的前CP(Apo-ceruloplasmin, Apo-CP)组成。研究发现,WD患者存在Apo-CP向Holo-CP转化障碍,血清中Holo-CP的表达水平较正常人明显减低。而血清CP水平正常的WD患者是否存在Apo-CP向Holo-CP的转化障碍,目前尚未见相关报道。
本课题基于多年来WD基因诊断的研究进展及研究者所在单位数十年来在WD诊疗方面的工作基础,采用限制性酶谱分析及DNA测序等技术寻找中国人WD患者新的突变热区和高频突变位点,为建立可用于临床的快速、简便的基因诊断方法奠定基础；收集晚发性WD患者和血清CP水平正常的WD患者的临床资料和基因组DNA标本,分析其临床特征和突变规律,检测WD患者血清中Holo-CP的表达水平,尝试探讨WD患者可能的分子发病机制。
实验目的
1.寻找中国人WD患者新的基因突变热区和高频突变位点,为建立可用于临床的快速、简便的基因诊断方法奠定基础。
2.研究中国人WD患者基因高频突变位点的基因型与临床表型的关系,探讨WD患者可能的分子发病机制。
3.研究中国人晚发性WD患者和血清CP水平正常的WD患者的临床特征和基因突变规律,为这两类特殊类型的WD患者的基因诊断奠定基础,探讨其可能的分子发病机制。
4.检测血清CP水平正常的WD患者血清Holo-CP的表达水平,结合其基因突变检测结果,探讨其可能的分子发病机制。
实验方法
本课题共包括四部分研究内容
第一章：WD基因突变热区和高频突变位点的检测
1.采用PCR-DNA测序技术检测来自华中和华东地区的139例非同源家系的WD患者ATP7B基因第13号外显子基因突变,以52名正常健康人为对照。
2.采用限制性酶谱分析技术检测142例来自不同地区的非同源家系的WD患者ATP7B基因第8、12、13外显子,以52名正常健康人为对照。
第二章：WD患者高频突变位点的基因型与临床表型关系的研究
以限制性酶谱分析法检测的142例WD患者为研究对象,收集上述患者的临床资料、基因检测结果,以第8、13号外显子Arg778Leu和Pro992Leu突变作为基因型,将患者的性别、发病年龄、病程、临床分型、首发症状及CP和血清铜氧化酶(serum copper oxidase, Sco)活性等铜生化检测结果等临床表型作为比较的指标,探讨中国人WD患者高频基因突变位点的基因型与临床表型的关系,在此基础上探讨WD患者可能的分子发病机制。
第三章：晚发性WD患和血清CP水平正常的WD患者的基因诊断及其临床特征分析
收集13例晚发性WD患者和19例血清CP水平正常的WD患者的临床资料和基因组DNA标本,总结这两类特殊类型WD患者的临床特征,采用限制性酶谱分析技术结合全长外显子DNA测序技术分析其基因突变规律,探讨这两类特殊类型WD患者可能的分子发病机制。
第四章：血清CP水平正常的WD患者血清Holo-CP表达水平的检测及其发病机制的探讨
收集了19例血清CP水平正常的WD患者的血清标本,以30例正常健康人和30例血清CP水平明显减低的典型WD患者为对照,采用非变性聚丙烯酰胺凝胶电泳(native polyacrylamide gel electrophoresis, native-PAGE)和Western blot技术检测WD患者和正常健康对照者血清Holo-CP(Holo-ceruloplasmin)的表达水平,结合其基因突变检测结果探讨可能的分子发病机制,尝试为本病患者提供一种较为敏感的蛋白标志物检测指标。
实验结果
第一章：WD基因突变热区和高频突变位点的检测
1.PCR-DNA测序结果显示,52例正常对照者未发现突变,WD患者共检出Arg969Gln、Ser975Tyr、Thr977Met、Gly988Val、Pro992Leu和Ala1018Val等6种突变类型和1种2866-13C>G多态,其中Gly988Val杂合突变为新的发现(GENEBANK注册号为FJ705811), Arg969Gln在中国人WD患者中首次发现,Arg969Gln合并Gly988Val复合杂合突变对应较重的临床表型。WD患者组突变及多态总的检出率为29.49%(41/139),染色体突变频率为16.19%(45/278)。Pro992Leu纯合/杂合突变的检出率为23.02%(32/139),染色体突变频率为12.59%(35/278)。
2.限制性酶谱分析结果显示,48.59%(69/142)的患者在第8外显子检出Arg778Leu突变,其中纯合突变13例,杂合突变56例；5.63%(8/142)的患者在第12外显子检出Thr935Met杂合突变；24.65%(35/142)的患者在第13外显子检出Pro992Leu突变,其中纯合突变3例,杂合突变32例。检出突变的患者中有12例为Arg778Leu/Pro992Leu复合杂合突变,1例Arg778Leu/Thr935Met复合杂合突变,1例Arg778Leu纯合突变合并Pro992Leu杂合突变,总检出率为69.01%(98/142)。以上结果均经DNA测序证实。
第二章：WD患者高频突变位点的基因型与临床表型关系的研究
1.142例WD患者中,Arg778Leu突变、Pro992Leu突变和Arg778Leu-Pro992Leu纯合/复合杂合突变与临床分型、首发症状、性别和病程等临床表型之间均无显著性差异(P>0.05)。
2.142例WD患者中,Arg778Leu纯合突变、Pro992Leu纯合突变、Arg778Leu-Pro992Leu纯合突变患者的发病年龄分别小于Arg778Leu杂合突变和无Arg778Leu突变、Pro992Leu杂合突变和无Pro992Leu突变、单个杂合突变和无Arg778Leu、Pro992Leu突变的患者,但各组间无显著性差异(P值分别为0.556、0.784、0.465)。
3.142例WD患者中,Arg778Leu纯合突变患者的血清Sco及CP水平低于Arg778Leu杂合突变和无Arg778Leu突变的患者,其中Sco水平无统计学意义(P值为0.058),CP水平有统计学意义(P值为0.038)。
4.142例WD患者中,Pro992Leu纯合突变患者的血清Sco及CP水平低于Pro992Leu杂合突变和无Pro992Leu突变的患者,但各组间无显著性差异(P值分别为0.432、0.300)。
5.142例WD患者中,Arg778Leu-Pro992Leu纯合突变、Arg778Leu-Pro992Leu复合杂合突变患者的血清Sco及CP水平显著低于单个杂合突变、无Arg778Leu突变和无Pro992Leu突变的患者(P值分别为0.033、0.011)。
第三章：晚发性WD患和血清CP正常的WD患者的基因诊断及其临床特征分析
1.13例晚发性WD患者中男性6例,女性7例；发病年龄40～60岁,平均(44.85±5.46)岁；病程0.3～8年,平均(2.61±2.21)年；肝型患者3例,脑型患者7例,脑-内脏型患者3例；以肝症状起病者6例,以锥体外系等神经系统症状起病者7例；有家族遗传病史者4例。血清铜、CP和Sco等铜生化检测结果均符合WD的改变,有5例以肝症状起病的患者入院时查肝功能结果异常；角膜K-F环阳性者12例,但有1例60岁发病者角膜K-F环为阴性；腹部超声检查结果显示患者均存在肝硬化改变,并伴有脾肿大、慢性胆囊炎等改变,脑型患者的头颅MRI等影像学检查示双侧基底节区异常信号。本组患者以神经系统症状起病者起病缓慢、肝损害症状较轻,驱铜治疗可获良好疗效；而以肝症状起病或伴发肝症状的患者出现明显的锥体外系症状和肝损害症状,病情相对较重,驱铜治疗后部分症状可获改善。
2.13例晚发性患者的ATP7B基因各外显子及启动子区共发现Gln511Stop、Arg778Leu、Arg919Gly、Pro992Leu、Ala1018Val、Arg1041Trp等6种形式的突变和-75A>C、-129_-125del、Ser406Ala、Va1456Leu、Leu770Leu、2448-25G>A、Lys832Arg、Lys952Arg、Val1106Ile、Ala1140Val、3903+6T>C等11种形式的多态,11例患者的基因突变均在第8、12、13外显子,且均为杂合突变或复合杂合突变。在检出的突变类型中,第8外显子Arg778Leu和第13外显子Pro992Leu杂合突变在本组患者中的检出率分别为46.15%(6/13)和30.77%(4/13),未检出第12外显子Thr935Met突变,但在第12外显子检出2例Arg919Gly杂合突变。
3.19例血清CP水平正常的WD患者中男性9例,女性10例；发病年龄4～31岁,平均(14.68±7.72)岁；病程0.1～15年,平均(3.89±4.09)年；肝型患者16例,脑型患者1例,脑-内脏型患者1例,症状前期的亚临床型患者1例；以肝症状起病者17例,以锥体外系等神经系统症状起病者1例,无症状者1例；有家族遗传病史者5例。血清铜、CP和Sco等铜生化检测结果正常或基本正常,有9例患者入院时查肝功能结果异常；角膜K-F环阳性者12例,角膜K-F环阴性者7例；腹部超声检查结果显示患者呈肝硬化或急性肝损伤的声像图改变；2例有神经系统症状的患者头颅影像学检查示双侧基底节区异常信号。有4例患者在外院行肝穿刺活检铜染色阳性,所有患者均行青霉胺负荷试验(penicillamine challenge test, PCT)确诊。
4.19例血清CP水平正常的WD患者ATP7B基因启动子及各外显子共发现11种形式的突变和14种形式的多态。其中,1870-65G>A、Met645Leu、2575+81A>G和Ala874Pro等4种突变为未曾报道的新的突变类型。本组19例患者中有16例患者检出突变,有3例患者未检出任何致病突变,其突变检出率为84.21%(16/19)。在检出突变的患者中,有3例患者为纯合突变,有2例患者每人存在2种杂合突变即复合杂合突变,其余检出突变的患者每人只存在1种杂合突变类型,突变形式以错义突变为主,但有3种剪接点突变。本组患者Arg778Leu的检出率为15.79%(3/19),未检出Thr935Met和Pro992Leu突变。
第四章：血清CP水平正常的WD患者血清Holo-CP表达水平的检测及其发病机制的探讨
1.正常健康人组和血清CP水平正常的WD患者组可见比较靠近的Apo-CP和Holo-CP 2条条带,血清CP水平明显减低的典型WD患者Apo-CP条带较为明显,Holo-CP条带较暗或缺失。
2.血清CP减低的WD患者组和CP正常的WD患者组Holo-CP的表达水平均低于正常健康人组,三组间有极显著性差异(F=207.544,P=0.000)。
结论
1.139例华中和华东地区WD患者ATP7B基因第13号外显子发现6种形式的突变和1种多态。
2.139例华中和华东地区WD患者ATP7B基因第13号外显子发现一种未曾报道的Gly988Val突变,并在中国人WD患者中首次发现Arg969Gln突变。Arg969Gln合并Gly988Val复合杂合突变对应较重的临床表型。
3.第13号外显子是中国人WD患者ATP7B基因的一个突变热区。
4.限制性酶谱分析技术检测WD基因第8、12、13号外显子能明显提高中国人WD患者基因突变的检出率,可用于临床对WD疑诊患者的快速筛选。
5.Pro992Leu突变是中国人WD患者的一个高频突变位点。
6.142例中国人WD患者Arg778Leu突变、Pro992Leu突变和Arg778Leu-Pro992Leu复合杂合突变与临床分型、首发症状、性别、病程和发病年龄等临床表型之间无相关性。
7.142例中国人WD患者Arg778Leu纯合突变、Pro992Leu纯合突变和Arg778Leu-Pro992Leu复合杂合突变的血清Sco及CP水平低于单个杂合突变和未检出Arg778Leu突变和Pro992Leu突变的患者。
8.Arg778Leu纯合突变和Pro992Leu纯合突变是对WD蛋白功能影响较大的致病性突变。
9.13例晚发性WD患者中以神经系统症状起病者主要表现锥体外系症状,肝损害症状较轻,驱铜治疗可获良好疗效。
10.13例晚发性WD患者中以肝症状起病者病情相对较重,并可伴有锥体外系症状,驱铜治疗后部分症状可获改善。
11.13例晚发性WD患者共发现6种突变类型和11种多态类型。
12.13例晚发性WD患者均为杂合突变或复合杂合突变,Arg778Leu杂合突变和Pro992Leu杂合突变检出率分别为46.15%和30.77%。
13.19例血清CP水平正常的WD患者以肝损伤症状为主要临床表现,但也有以脑症状起病或亚临床型的无症状患者。
14.19例血清CP水平正常的WD患者共发现11种形式的突变和14种形式的多态。
15.19例血清CP水平正常的WD患者Arg778Leu突变的检出率为15.79%,未检出Thr935Met和Pro992Leu突变。
16.19例血清CP水平正常的WD患者发现4种未曾报道的新突变类型。
17.采用native-PAGE和Western blot检测结果显示,血清CP减低的典型WD患者血清Holo-CP的表达水平明显减低,存在Apo-CP向Holo-CP的转化障碍。
18.采用native-PAGE和Western blot检测结果显示,血清CP水平正常的WD患者血清Holo-CP的表达水平显著低于健康对照者(P=0.013),Apo-CP向Holo-CP的转化障碍也是其重要发病原因。对于临床难以诊断的WD疑诊患者,采用native-PAGE和Western blot检测Holo-CP的表达水平是一种较为敏感的诊断指标。
本课题的主要创新点
1.139例华中和华东地区WD患者ATP7B基因第13号外显子发现一种未曾报道的Gly988Val突变,并在中国人WD患者中首次发现Arg969Gln突变。
2.第13号外显子是中国人WD患者ATP7B基因的一个突变热区。
3.Pro992Leu突变是中国人WD患者的一个高频突变位点。
4.142例中国人WD患者Arg778Leu纯合突变、Pro992Leu纯合突变和Arg778Leu-Pro992Leu复合杂合突变的血清Sco及CP水平低于单个杂合突变和未检出Arg778Leu突变和Pro992Leu突变的患者,Arg778Leu纯合突变和Pro992Leu纯合突变是对WD蛋白功能影响较大的致病性突变。
5.13例晚发性WD患者共发现6种突变类型和11种多态类型,均为杂合突变或复合杂合突变,Arg778Leu杂合突变和Pro992Leu杂合突变检出率分别为46.15%和30.77%。
6.19例血清CP水平正常的WD患者以肝损伤症状为主要临床表现,但也有以脑症状起病或亚临床型的无症状患者。
7.19例血清CP水平正常的WD患者共发现11种形式的突变和14种形式的多态,并发现4种未曾报道的新突变类型。
8.采用native-PAGE和Western blot检测结果显示,血清CP水平正常的WD患者血清Holo-CP的表达水平低于正常对照者,也存在Apo-CP向Holo-CP的转化障碍,对于临床难以诊断的WD疑诊患者,采用native-PAGE和Westernblot检测Holo-CP的表达水平是一种较为敏感的诊断指标。
Background
Hepatolenticular degeneration (HLD), also called Wilson disease (WD), is an autosomal recessively recessively inherited disorder of copper metabolism, with an incidence of 15～30 in 1,000,000 in different populations. WD gene, which located in 13q14.3, encoded a 165Kda copper-transporting P-type ATPase (ATP7B) containing 1465 amino acids, and also called ATP7B gene. ATP7B gene mutations can lead to functional deficit of ATP7Base in hepatocytes, which located in trans-Golgi network (TGN) and membrane of bile vessel, and result in transmembrane transport disturbance of copper ion, dyssynthesis of ceruloplasmin (CP) and impaired biliary excretion of copper in hepatocytes. Finally, excess toxic copper accumulate predominantly in the liver, brain, kidney and comea, and caused lower serum CP levels, cirrhosis, neurological/psychiatric symptoms and Kayser-Fleischer ring (K-F ring) in WD patients.
WD is a treatable inherited neurological disease. Most of WD patients can obtain eusemia if they accepted therapy in time. Otherwise, their conditions would be worse or even died. However, many WD patients are often misdiagnosed as other diseases because their early symptoms are complicated and variable and the laboratory copper metabolism tests are often acquired false positive or false negative results in some patients. So it is difficult to make early diagnosis, especially presymptomatic diagnosis and antenatal diagnosis for WD patients before WD gene was cloned.
Many data showed that WD gene mutations were highly genetic heterogeneity. There are more than 300 mutations were found in WD patients and most of them are compound heterozygous mutations. The common mutation types are rare and most of them are scarce mutations. The features of mutations are obviously distinct in different populations. The highly genetic heterogeneity can explain the reasons of complicated and variable symptoms in these patients, but it takes many difficulties for gene diagnosis of WD patients. For this reason, it is a fast and high-performance gene diagnosis strategy for WD patients that identification hot regions of mutations in different ethnic populations, and using techniques such as PCR or multiplex PCR to amplify these regions and scan these regions by sequencing.
Because copper deposition in the liver is usually experienced accumulation stage and saturation stage for WD patients, most of them would be onset between 5 and 35 years. However, recent epidemiological investigations showed that not a few patients present illness after 40 years old. These patients are not typically and can be misdiagnosed as other diseases more often. For WD patients, low serum CP level is a distinctive biochemistry change, and it is the most important evidence for diagnosis too. However, there are 5 percent WD patients with normal CP levels. These 2 special types of WD patients are more often misdiagnosed as other diseases, and gene diagnosis can provide more powerful evidences for them. But few research data was found for these patients.
Sera CP in the body are combined with Holo-CP (conjugated with copper ion) and Apo-CP (not conjugated with copper ion). Recent research showed that transform disturbance from Apo-CP to Holo-CP in WD patients, and their sera Holo-CP expression levels were decreased compared with normal heath person. There have been no report about Holo-CP expression levels in WD patients with normal CP levels from now on.
Based on the progress of gene diagnosis for WD and the foundation of our research on diagnosis and treatment for WD during past decades years, this project plan to find out new hot regions or high frequency spots of gene mutations for Chinese WD patients by restriction mapping analysis and DNA sequencing, and attempt to establish a quick and convenient gene diagnosis method for clinical practice. At the same time, this project plan to collect clinical data and genomic DNA samples of WD patients with late onset or with normal sera CP levels, analyze their clinical features and characters of gene mutations, detect their sera Holo-CP expression levels and attempt to study their molecular pathogenesis.
Objeetives
1. To find out new hot regions or high frequency spots of gene mutations for Chinese WD patients, attempt to establish a quick and convenient gene diagnosis method for clinical practice.
2. To analyze the relationships between the genotypes of high frequency mutation spots of Chinese WD patients and their clinical phenotypes, attempt to study their molecular pathogenesis.
3. To analyze clinical features and characters of gene mutations of Chinese WD patients with late onset or with normal sera CP levels, attempt to study their molecular pathogenesis.
4. To detect sera Holo-CP expression levels of WD patients with normal CP levels, attempt to study their molecular pathogenesis and provide a more sensitive protein marker for them.
Methods
The experiment was composed of four parts.
Chapter 1:Detection for hot regions or high frequency spots of gene mutations of WD.
1. Exon 13 of ATP7B gene in 139 unrelated WD patients which came from middle and eastern China and 52 unrelated normal controls were detected by PCR-DNA sequencing.
2. Exon8,12,13 of ATP7B gene in 142 unrelated WD patients and 52 unrelated normal controls were detected by restriction mapping analysis.
Chapter 2:The relationships between the genotypes of high frequency mutation spots and clinical phenotypes in patients with WD.
The clinical data and results of gene mutations of 142 WD patients which were detected by restriction mapping analysis were collected. Arg778Leu of exon8 and Pro992Leu mutations of exon13 were as the genotypes, and clinical types, gender, age of onset, courses of disease, first symptoms and the detection results of copper metabolism, such as CP and serum copper oxidase (Sco)were as clinical phenotypes. The relationships between the genotypes of high frequency mutation spots of Chinese WD patients and their clinical phenotypes were analyzed.
Chapter 3:Gene diagnosis and the clinical features of WD patients with late-onset and normal sera CP levels.
The clinical data and genomic DNA samples of 13 cases of WD patients with late-onset and 19 cases of WD patients with normal sera CP levels were collected. Their clinical features were analyzed and their mutations were detected by restriction mapping analysis and DNA sequencing including promoter and all exons.
Chapter 4:Detection of sera Holo-CP expression levels of WD patients with normal CP levels and study on their pathogenesis.
Sera samples were collected in 19 WD patients with normal CP levels,30 normal controls and 30 WD patients with lower CP levels. Holo-CP expression levels were detected by native polyacrylamide gel electrophoresis (native-PAGE) and Western blot.
Results
ChapterⅠ:Detection for hot regions or high frequency spots of gene mutations of WD.
1. No abnormality was founded in 52 controls. In 139 patients,6 mutations and 1 polymorphism were found, including 1 novel mutation (Gly988Val). The novel mutation was deposited in GeneBank with the Accession Numbers FJ705811. The compound heterozygous mutation of Arg969Gln/Gly988Val corresponds to severious clinical phenotype. The rate of mutations and polymorphisms of Exon13 was 29.49% (41/139), and its frequency of chromosome mutation was 16.19%(45/278). The rate of Pro992Leu homozygous/heterozygous mutations was 23.02%(32/139), and its frequency of chromosome mutation was 12.59%(35/278).
2. Arg778Leu of exon8 was detected in 48.59%(69/142) patients.13 patients were homozygous mutation and another 56 patients were heterozygous mutation. Thr935Met of exonl2 was detected in 5.63%(8/142) patients and 8 cases were all heterozygous mutation. Pro992Leu of exonl3 was detected in 24.65%(35/142) patients.3 patients were homozygous mutation and 32 patients were heterozygous mutation. Among them,12 cases were compound heterozygous mutation with Arg778Leu/Pro992Leu,1 case was compound heterozygous mutation with Arg778Leu/Thr935Met, and 1 case was Arg778Leu homozygous mutation which combined with Pro992Leu heterozygous mutation. Mutations were detected in 69.01% (98/142) patients totally. The results of this method were confirmed by DNA sequencing.
Chapter 2:The relationships between the genotypes of high frequency mutation spots and clinical phenotypes in patients with WD.
1. There were no relationships between the genotypes of Arg778Leu mutation, Pro992Leu mutation and their clinical phenotypes of clinical types, gender, first symptoms, courses of disease and the levels of CS (P>0.05).
2. The average ages of onset of Arg778Leu homozygous mutation group, Pro992Leu homozygous mutation group and Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than heterozygous mutation group and no Arg778Leu or Pro992Leu mutation group, but there were no statistical significance(P>0.05).
3. The levels of Sco and CP of Arg778Leu homozygous mutation group were lower than no Arg778Leu mutation group significantly (P=0.019, and P=0.011). The levels of Sco and CP of Arg778Leu homozygous mutation group were lower than Arg778Leu heterozygous mutation group, the levels of Sco was no statistical significance(P=0.081) and the levels of CP was statistical significance(P=0.042). The levels of Sco and CP of Arg778Leu heterozygous mutation group were lower than no Arg778Leu mutation group, but no statistical significance(P=0.328, and P= 0.408).
4. The levels of Sco and CP of Pro992Leu homozygous mutation group and Pro992Leu heterozygous mutation group were lower than no Pro992Leu mutation group, and the levels of Sco and CP of Pro992Leu homozygous mutation group were lower than Pro992Leu heterozygous mutation group, but there were all no statistical significance (P>0.05).
5. The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group and Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than no Arg778Leu and no Pro992Leu mutation group significantly (P values were 0.011 and 0.049 for Sco, P values were 0.003 and 0.055 for CP). The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group were lower than only Arg778Leu or Pro992Leu heterozygous group significantly (P value was 0.049 for Sco, P value was 0.009 for CP). The levels of Sco and CP of Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than only Arg778Leu or Pro992Leu heterozygous group, but there were no statistical significance (P>0.05). The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group were lower than Arg778Leu-Pro992Leu compound heterozygous mutation group, but there were no statistical significance (P>0.05).
Chapter 3:Gene diagnosis and the clinical features of WD patients with late-onset and normal sera CP levels.
1. In 13 WD patients with late-onset,6 cases were male and 7 cases were female. Ages of onset were from 40 to 60 years, and their average age of onset was (2.61±2.21) years. Courses of disease were from 0.3 to 8 years and their average course of disease was (2.61±2.21) years.3 cases were patients with hepatic type,7 cases were patients with cerebral type and 3 cases were patients with cerebral-visceral type.6 cases were onset with hepatic symptoms,7 cases were onset with neurological symptoms. Their detection results of Scu, CP and SC were consistent with WD, and 5 of them had abnormal detection results of liver function. They all had comea K-F rings except 1 patient which age of onset was 60 years. The results of abdominal ultrasound examination showed that they all were cirrhosis. Cranium MRI showed abnormal signal in basal ganglia. Compared with patients who were onset with hepatic symptoms, the patients who were onset with neurological symptoms were slow onset and their conditions were less severiouly.
2. There were 6 mutations and 11 polymorphisms in promoter region and all exons of ATP7B gene for these 13 WD patients with late-onset, and 11 cases of them had mutations in exon8,12 and 13. They were all heterozygous mutations or combined heterozygous mutations. The frequency of Arg778Leu was 46.15%(6/13) and Pro992Leu was 30.77%(4/13) in these WD patients. Thr935Met mutation was not found, but 2 cases were detected Arg919Gly heterozygous mutation.
3. In 19 WD patients with normal sera CP levels,9 cases were male and 10 cass were female. Ages of onset were from 4 to 31 years and their average age of onset was (14.68±7.72) years. Courses of disease were from 0.1 to 15 years and their average courses of disease was (3.89±4.09) years.16 cases were patients with hepatic type,1 case was patient with cerebral type,1 case was patient with cerebral-visceral type and 1 case was patient with presymptomatic type.17 cases were onset with hepatic symptoms,2 cases were onset with neurological symptoms. Their detection results of Sco, CP and SC were normal, and 9 of them had abnormal detection results. 12 cases of them had comea K-F rings. The results of abdominal ultrasound examination showed that they all were cirrhosis or acute hepatic injury. Cranium MRI showed abnormal signal in basal ganglia in 2 cases of patients with neurological symptoms.4 cases of them were diagnosed by liver biopsy, and all patients were had final diagnosis by penicillamine challenge test (PCT).
4. There were 11 mutations and 14 polymorphisms in promoter region and all exons of ATP7B gene for these 19 WD patients with normal CP levels.4 mutations including 1870-65G>A, Met645Leu,2575+81A>G and Ala874Pro were novel. In these 19 WD patients,16 cases of them were detected mutations, and any mutations were not found in 3 cases of them. The rate of mutations detection was 84.21% (16/19)。For 16 patients who were detected mutations,3 cases were homozygous mutation,2 cases were compound heterozygous mutation, and only 1 mutation were found in the others cases. The missense mutations is the major mutation types, and 3 kinds of splice junction mutations were found. The frequency of Arg778Leu was 15.79% (3/19), Thr935Met and Pro992Leu mutation were not found.
Chapter 4:Detection of sera Holo-CP expression levels of WD patients with normal CP levels and study on their pathogenesis.
The expression levels of sera Holo-CP of normal controls and WD patients with normal CP levels were higher than WD patients with low CP levels (P=0.000), and the expression levels of sera Holo-CP of WD patients with normal CP levels was lower than normal controls (P=0.013).
Conclusions
1.6 mutations and 1 polymorphism were found in ATP7B gene of 139 WD patients which came from middle and eastern China.
2. Gly988Val, which was a novel mutation, was found in ATP7B gene of 139 WD patients which came from middle and eastern China. Arg969Gln mutation was found first time in Chinese WD patients. The compound heterozygous mutation of Arg969Gln/Gly988Val corresponds to severious clinical phenotype.
3. Exon 13 of ATP7B gene is one of hot mutation regions in Chinese WD patients.
4. The detection rates of gene mutations can be significantly improved by analysis exon8,12 and 13 of WD gene with the method of restriction mapping. The method can help to diagnose suspicious WD cases and their family members in clinical.
5. Pro992Leu mutation is one of high frequency mutation spots in Chinese WD patients.
6. There were no relationships between the genotypes of Arg778Leu mutation, Pro992Leu mutation, Arg778Leu-Pro992Leu compound heterozygous mutation and the clinical phenotypes of clinical types, first symptoms, genders, courses of disease and average ages of onset in 142 Chinese WD patients..
7. The levels of Sco and CP of WD patients with Arg778Leu homozygous mutation, Pro992Leu homozygous mutation, Arg778Leu-Pro992Leu homozygous/ compound heterozygous mutation were lower than those with no Arg778Leu mutation or no Pro992Leu mutation and only Arg778Leu mutation mutation or only Pro992Leu heterozygous mutation.
8. Arg778Leu mutationand Pro992Leu mutation are pathogenic mutations which affected the function of WD protein seriously.
9. Extrapyramidal symptoms were mainly clinical manifestations in 13 WD patients with late-onset which were onset with neurological symptoms. Their liver damage was mild and good effect can be acquired for them by de-copper treatment.
10. Compared with WD patients with late-onset which were onset with neurological symptoms. The patients which were onset with liver symptoms were serious condition. Some of them were accompanied with extrapyramidal symptoms and parts of symptoms can be improved after de-copper treatment.
11.6 mutations and 11 polymorphisms were found in ATP7B gene of 13 WD patients with late-onset.
12. The mutations of 13 WD patients with late-onset were all heterozygous mutations or compound heterozygous mutations, the detection rates of mutations were 46.15% for Arg778Leu heterozygous mutation and 30.77% for Pro992Leu heterozygous mutation in these patients.
13. Liver damage symptoms were mainly clinical manifestations in 19 WD patients with normal CP levels. However, there were patients who were onset with neurological symptoms or without any symptoms.
14.11 mutations and 14 polymorphisms were found in ATP7B gene of 19 WD patients with normal CP levels.
15. The detection rate of Arg778Leu mutation was 15.79% in 19 WD with normal CP levels. Thr935Met mutation and Pro992Leu mutation were not found in these patients. The total detection rate of mutations in exon8,12 and 13 was 42.11%.
16.4 novel mutations were found in ATP7B gene of 19 WD patients with normal CP levels.
17. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in typical WD patients with low CP levels by native-PAGE and Western blot.
18. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in WD patients with mormal CP levels by native-PAGE and Western blot.The detection of Holo-CP expression levels is a sensitive diadynamic criteria for skeptical WD patients in clinical practice.
The originalities of this current project lie in:
1. Gly988Val, which was a novel mutation, was found in ATP7B gene of 139 WD patients which came from middle and eastern China. Arg969Gln mutation was found first time in Chinese WD patients.
2. Exon 13 of ATP7B gene is one of hot mutation regions in Chinese WD patients.
3. Pro992Leu mutation is one of high frequency mutation spots in Chinese WD patients.
4. The levels of Sco and CP of WD patients with Arg778Leu homozygous mutation, Pro992Leu homozygous mutation, Arg778Leu-Pro992Leu homozygous/ compound heterozygous mutation were lower than those with no Arg778Leu mutation or no Pro992Leu mutation and only Arg778Leu mutation mutation or only Pro992Leu heterozygous mutation.
5.6 mutations and 11 polymorphisms were found in ATP7B gene of 13 WD patients with late-onset. These mutations were all heterozygous mutations or compound heterozygous mutations, the detection rates of mutations were 46.15% for Arg778Leu heterozygous mutation and 30.77% for Pro992Leu heterozygous mutation.
6. Liver damage symptoms were mainly clinical manifestations in 19 WD patients with normal CP levels. However, there were patients who were onset with neurological symptoms or without any symptoms.
7.11 mutations and 14 polymorphisms were found in ATP7B gene of 19 WD patients with normal CP levels.4 novel mutations were found in ATP7B gene of these WD patients.
8. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in WD patients with mormal CP levels by native-PAGE and Western blot.The detection of Holo-CP expression levels is a sensitive diadynamic criteria for skeptical WD patients in clinical practice.
肝豆状核变性(Hepatolenticular degeneration, HLD)又名Wilson病(Wilson's disease, WD),是一种常染色体隐性遗传性铜代谢障碍疾病,不同人群的发病率约为15～30/100万,其基因定位于13q14.3, cDNA编码由1465个氨基酸组成的分子量约165Kda的一种P型铜转运ATP酶(copper-transporting P-type ATPase, ATP7B)。WD患者由于ATP7B基因突变使位于肝细胞高尔基反面网状结构(trans-Golgi network, TGN)及胆管膜侧的ATP7B酶功能缺陷,引起铜离子跨膜转运障碍,导致铜蓝蛋白(ceruloplasmin, CP)合成障碍及胆汁中铜的排泄受限,过量的铜不能排泄出体外而在肝、脑、肾、角膜等部位沉积并引起相应的组织脏器损伤,患者可出现血清CP水平减低、肝硬化、神经/精神症状、角膜K-F环等临床表现。
WD是少数可以治疗的神经遗传病之一,患者如果能在发病早期或症状前期被确诊并得到及时治疗,大多预后良好,反之病情逐渐加重甚至危及生命。但由于铜在不同脏器中沉积的速度和程度不同,许多患者早期症状复杂多样,极易被误诊为其他疾病,实验室铜代谢检查等又存在假阳性或假阴性结果,因此本病的早期诊断特别是症状前期和产前诊断较为困难。WD基因的克隆,使本病患者通过基因诊断技术进行症状前期及产前诊断成为可能。
大量研究结果证实,WD基因存在高度的遗传异质性,各外显子的突变位点众多,目前已发现超过300种形式的基因突变,且多为复合杂合突变,常见突变形式少见,多为罕见突变,在不同人种的突变特征亦存在明显差异。这虽然解释了WD患者临床表现复杂多样的原因,但给基因诊断走向临床实用带来了困难。因此,明确不同地域、不同人群WD基因的突变热区和高频突变位点并采用PCR或多重PCR技术扩增相应人群的突变热区并进行序列比对分析,是检出WD患者的一种快速、高效的基因诊断策略。
由于WD患者肝脏中铜的沉积通常经历肝铜蓄积期、肝铜饱和释放期等病理过程,大多在5～35岁之间起病。但近来的多个流行病学调查研究发现,40岁以上起病的晚发性WD患者并不少见。血清CP水平减低是WD的特征性生化改变,也是最主要的诊断依据,但约有5%左右的WD患者血清CP水平在正常范围。由于这两种特殊类型的WD患者在临床中极易被误诊误治,常给临床医生的WD诊疗工作带来困难,而基因诊断可为其提供最有力的诊断依据。
血清CP由与铜离子结合的全CP(Holo-ceruloplasmin, Holo-CP)和未与铜离子结合的前CP(Apo-ceruloplasmin, Apo-CP)组成。研究发现,WD患者存在Apo-CP向Holo-CP转化障碍,血清中Holo-CP的表达水平较正常人明显减低。而血清CP水平正常的WD患者是否存在Apo-CP向Holo-CP的转化障碍,目前尚未见相关报道。
本课题基于多年来WD基因诊断的研究进展及研究者所在单位数十年来在WD诊疗方面的工作基础,采用限制性酶谱分析及DNA测序等技术寻找中国人WD患者新的突变热区和高频突变位点,为建立可用于临床的快速、简便的基因诊断方法奠定基础；收集晚发性WD患者和血清CP水平正常的WD患者的临床资料和基因组DNA标本,分析其临床特征和突变规律,检测WD患者血清中Holo-CP的表达水平,尝试探讨WD患者可能的分子发病机制。
实验目的
1.寻找中国人WD患者新的基因突变热区和高频突变位点,为建立可用于临床的快速、简便的基因诊断方法奠定基础。
2.研究中国人WD患者基因高频突变位点的基因型与临床表型的关系,探讨WD患者可能的分子发病机制。
3.研究中国人晚发性WD患者和血清CP水平正常的WD患者的临床特征和基因突变规律,为这两类特殊类型的WD患者的基因诊断奠定基础,探讨其可能的分子发病机制。
4.检测血清CP水平正常的WD患者血清Holo-CP的表达水平,结合其基因突变检测结果,探讨其可能的分子发病机制。
实验方法
本课题共包括四部分研究内容
第一章：WD基因突变热区和高频突变位点的检测
1.采用PCR-DNA测序技术检测来自华中和华东地区的139例非同源家系的WD患者ATP7B基因第13号外显子基因突变,以52名正常健康人为对照。
2.采用限制性酶谱分析技术检测142例来自不同地区的非同源家系的WD患者ATP7B基因第8、12、13外显子,以52名正常健康人为对照。
第二章：WD患者高频突变位点的基因型与临床表型关系的研究
以限制性酶谱分析法检测的142例WD患者为研究对象,收集上述患者的临床资料、基因检测结果,以第8、13号外显子Arg778Leu和Pro992Leu突变作为基因型,将患者的性别、发病年龄、病程、临床分型、首发症状及CP和血清铜氧化酶(serum copper oxidase, Sco)活性等铜生化检测结果等临床表型作为比较的指标,探讨中国人WD患者高频基因突变位点的基因型与临床表型的关系,在此基础上探讨WD患者可能的分子发病机制。
第三章：晚发性WD患和血清CP水平正常的WD患者的基因诊断及其临床特征分析
收集13例晚发性WD患者和19例血清CP水平正常的WD患者的临床资料和基因组DNA标本,总结这两类特殊类型WD患者的临床特征,采用限制性酶谱分析技术结合全长外显子DNA测序技术分析其基因突变规律,探讨这两类特殊类型WD患者可能的分子发病机制。
第四章：血清CP水平正常的WD患者血清Holo-CP表达水平的检测及其发病机制的探讨
收集了19例血清CP水平正常的WD患者的血清标本,以30例正常健康人和30例血清CP水平明显减低的典型WD患者为对照,采用非变性聚丙烯酰胺凝胶电泳(native polyacrylamide gel electrophoresis, native-PAGE)和Western blot技术检测WD患者和正常健康对照者血清Holo-CP(Holo-ceruloplasmin)的表达水平,结合其基因突变检测结果探讨可能的分子发病机制,尝试为本病患者提供一种较为敏感的蛋白标志物检测指标。
实验结果
第一章：WD基因突变热区和高频突变位点的检测
1.PCR-DNA测序结果显示,52例正常对照者未发现突变,WD患者共检出Arg969Gln、Ser975Tyr、Thr977Met、Gly988Val、Pro992Leu和Ala1018Val等6种突变类型和1种2866-13C>G多态,其中Gly988Val杂合突变为新的发现(GENEBANK注册号为FJ705811), Arg969Gln在中国人WD患者中首次发现,Arg969Gln合并Gly988Val复合杂合突变对应较重的临床表型。WD患者组突变及多态总的检出率为29.49%(41/139),染色体突变频率为16.19%(45/278)。Pro992Leu纯合/杂合突变的检出率为23.02%(32/139),染色体突变频率为12.59%(35/278)。
2.限制性酶谱分析结果显示,48.59%(69/142)的患者在第8外显子检出Arg778Leu突变,其中纯合突变13例,杂合突变56例；5.63%(8/142)的患者在第12外显子检出Thr935Met杂合突变；24.65%(35/142)的患者在第13外显子检出Pro992Leu突变,其中纯合突变3例,杂合突变32例。检出突变的患者中有12例为Arg778Leu/Pro992Leu复合杂合突变,1例Arg778Leu/Thr935Met复合杂合突变,1例Arg778Leu纯合突变合并Pro992Leu杂合突变,总检出率为69.01%(98/142)。以上结果均经DNA测序证实。
第二章：WD患者高频突变位点的基因型与临床表型关系的研究
1.142例WD患者中,Arg778Leu突变、Pro992Leu突变和Arg778Leu-Pro992Leu纯合/复合杂合突变与临床分型、首发症状、性别和病程等临床表型之间均无显著性差异(P>0.05)。
2.142例WD患者中,Arg778Leu纯合突变、Pro992Leu纯合突变、Arg778Leu-Pro992Leu纯合突变患者的发病年龄分别小于Arg778Leu杂合突变和无Arg778Leu突变、Pro992Leu杂合突变和无Pro992Leu突变、单个杂合突变和无Arg778Leu、Pro992Leu突变的患者,但各组间无显著性差异(P值分别为0.556、0.784、0.465)。
3.142例WD患者中,Arg778Leu纯合突变患者的血清Sco及CP水平低于Arg778Leu杂合突变和无Arg778Leu突变的患者,其中Sco水平无统计学意义(P值为0.058),CP水平有统计学意义(P值为0.038)。
4.142例WD患者中,Pro992Leu纯合突变患者的血清Sco及CP水平低于Pro992Leu杂合突变和无Pro992Leu突变的患者,但各组间无显著性差异(P值分别为0.432、0.300)。
5.142例WD患者中,Arg778Leu-Pro992Leu纯合突变、Arg778Leu-Pro992Leu复合杂合突变患者的血清Sco及CP水平显著低于单个杂合突变、无Arg778Leu突变和无Pro992Leu突变的患者(P值分别为0.033、0.011)。
第三章：晚发性WD患和血清CP正常的WD患者的基因诊断及其临床特征分析
1.13例晚发性WD患者中男性6例,女性7例；发病年龄40～60岁,平均(44.85±5.46)岁；病程0.3～8年,平均(2.61±2.21)年；肝型患者3例,脑型患者7例,脑-内脏型患者3例；以肝症状起病者6例,以锥体外系等神经系统症状起病者7例；有家族遗传病史者4例。血清铜、CP和Sco等铜生化检测结果均符合WD的改变,有5例以肝症状起病的患者入院时查肝功能结果异常；角膜K-F环阳性者12例,但有1例60岁发病者角膜K-F环为阴性；腹部超声检查结果显示患者均存在肝硬化改变,并伴有脾肿大、慢性胆囊炎等改变,脑型患者的头颅MRI等影像学检查示双侧基底节区异常信号。本组患者以神经系统症状起病者起病缓慢、肝损害症状较轻,驱铜治疗可获良好疗效；而以肝症状起病或伴发肝症状的患者出现明显的锥体外系症状和肝损害症状,病情相对较重,驱铜治疗后部分症状可获改善。
2.13例晚发性患者的ATP7B基因各外显子及启动子区共发现Gln511Stop、Arg778Leu、Arg919Gly、Pro992Leu、Ala1018Val、Arg1041Trp等6种形式的突变和-75A>C、-129_-125del、Ser406Ala、Va1456Leu、Leu770Leu、2448-25G>A、Lys832Arg、Lys952Arg、Val1106Ile、Ala1140Val、3903+6T>C等11种形式的多态,11例患者的基因突变均在第8、12、13外显子,且均为杂合突变或复合杂合突变。在检出的突变类型中,第8外显子Arg778Leu和第13外显子Pro992Leu杂合突变在本组患者中的检出率分别为46.15%(6/13)和30.77%(4/13),未检出第12外显子Thr935Met突变,但在第12外显子检出2例Arg919Gly杂合突变。
3.19例血清CP水平正常的WD患者中男性9例,女性10例；发病年龄4～31岁,平均(14.68±7.72)岁；病程0.1～15年,平均(3.89±4.09)年；肝型患者16例,脑型患者1例,脑-内脏型患者1例,症状前期的亚临床型患者1例；以肝症状起病者17例,以锥体外系等神经系统症状起病者1例,无症状者1例；有家族遗传病史者5例。血清铜、CP和Sco等铜生化检测结果正常或基本正常,有9例患者入院时查肝功能结果异常；角膜K-F环阳性者12例,角膜K-F环阴性者7例；腹部超声检查结果显示患者呈肝硬化或急性肝损伤的声像图改变；2例有神经系统症状的患者头颅影像学检查示双侧基底节区异常信号。有4例患者在外院行肝穿刺活检铜染色阳性,所有患者均行青霉胺负荷试验(penicillamine challenge test, PCT)确诊。
4.19例血清CP水平正常的WD患者ATP7B基因启动子及各外显子共发现11种形式的突变和14种形式的多态。其中,1870-65G>A、Met645Leu、2575+81A>G和Ala874Pro等4种突变为未曾报道的新的突变类型。本组19例患者中有16例患者检出突变,有3例患者未检出任何致病突变,其突变检出率为84.21%(16/19)。在检出突变的患者中,有3例患者为纯合突变,有2例患者每人存在2种杂合突变即复合杂合突变,其余检出突变的患者每人只存在1种杂合突变类型,突变形式以错义突变为主,但有3种剪接点突变。本组患者Arg778Leu的检出率为15.79%(3/19),未检出Thr935Met和Pro992Leu突变。
第四章：血清CP水平正常的WD患者血清Holo-CP表达水平的检测及其发病机制的探讨
1.正常健康人组和血清CP水平正常的WD患者组可见比较靠近的Apo-CP和Holo-CP 2条条带,血清CP水平明显减低的典型WD患者Apo-CP条带较为明显,Holo-CP条带较暗或缺失。
2.血清CP减低的WD患者组和CP正常的WD患者组Holo-CP的表达水平均低于正常健康人组,三组间有极显著性差异(F=207.544,P=0.000)。
结论
1.139例华中和华东地区WD患者ATP7B基因第13号外显子发现6种形式的突变和1种多态。
2.139例华中和华东地区WD患者ATP7B基因第13号外显子发现一种未曾报道的Gly988Val突变,并在中国人WD患者中首次发现Arg969Gln突变。Arg969Gln合并Gly988Val复合杂合突变对应较重的临床表型。
3.第13号外显子是中国人WD患者ATP7B基因的一个突变热区。
4.限制性酶谱分析技术检测WD基因第8、12、13号外显子能明显提高中国人WD患者基因突变的检出率,可用于临床对WD疑诊患者的快速筛选。
5.Pro992Leu突变是中国人WD患者的一个高频突变位点。
6.142例中国人WD患者Arg778Leu突变、Pro992Leu突变和Arg778Leu-Pro992Leu复合杂合突变与临床分型、首发症状、性别、病程和发病年龄等临床表型之间无相关性。
7.142例中国人WD患者Arg778Leu纯合突变、Pro992Leu纯合突变和Arg778Leu-Pro992Leu复合杂合突变的血清Sco及CP水平低于单个杂合突变和未检出Arg778Leu突变和Pro992Leu突变的患者。
8.Arg778Leu纯合突变和Pro992Leu纯合突变是对WD蛋白功能影响较大的致病性突变。
9.13例晚发性WD患者中以神经系统症状起病者主要表现锥体外系症状,肝损害症状较轻,驱铜治疗可获良好疗效。
10.13例晚发性WD患者中以肝症状起病者病情相对较重,并可伴有锥体外系症状,驱铜治疗后部分症状可获改善。
11.13例晚发性WD患者共发现6种突变类型和11种多态类型。
12.13例晚发性WD患者均为杂合突变或复合杂合突变,Arg778Leu杂合突变和Pro992Leu杂合突变检出率分别为46.15%和30.77%。
13.19例血清CP水平正常的WD患者以肝损伤症状为主要临床表现,但也有以脑症状起病或亚临床型的无症状患者。
14.19例血清CP水平正常的WD患者共发现11种形式的突变和14种形式的多态。
15.19例血清CP水平正常的WD患者Arg778Leu突变的检出率为15.79%,未检出Thr935Met和Pro992Leu突变。
16.19例血清CP水平正常的WD患者发现4种未曾报道的新突变类型。
17.采用native-PAGE和Western blot检测结果显示,血清CP减低的典型WD患者血清Holo-CP的表达水平明显减低,存在Apo-CP向Holo-CP的转化障碍。
18.采用native-PAGE和Western blot检测结果显示,血清CP水平正常的WD患者血清Holo-CP的表达水平显著低于健康对照者(P=0.013),Apo-CP向Holo-CP的转化障碍也是其重要发病原因。对于临床难以诊断的WD疑诊患者,采用native-PAGE和Western blot检测Holo-CP的表达水平是一种较为敏感的诊断指标。
本课题的主要创新点
1.139例华中和华东地区WD患者ATP7B基因第13号外显子发现一种未曾报道的Gly988Val突变,并在中国人WD患者中首次发现Arg969Gln突变。
2.第13号外显子是中国人WD患者ATP7B基因的一个突变热区。
3.Pro992Leu突变是中国人WD患者的一个高频突变位点。
4.142例中国人WD患者Arg778Leu纯合突变、Pro992Leu纯合突变和Arg778Leu-Pro992Leu复合杂合突变的血清Sco及CP水平低于单个杂合突变和未检出Arg778Leu突变和Pro992Leu突变的患者,Arg778Leu纯合突变和Pro992Leu纯合突变是对WD蛋白功能影响较大的致病性突变。
5.13例晚发性WD患者共发现6种突变类型和11种多态类型,均为杂合突变或复合杂合突变,Arg778Leu杂合突变和Pro992Leu杂合突变检出率分别为46.15%和30.77%。
6.19例血清CP水平正常的WD患者以肝损伤症状为主要临床表现,但也有以脑症状起病或亚临床型的无症状患者。
7.19例血清CP水平正常的WD患者共发现11种形式的突变和14种形式的多态,并发现4种未曾报道的新突变类型。
8.采用native-PAGE和Western blot检测结果显示,血清CP水平正常的WD患者血清Holo-CP的表达水平低于正常对照者,也存在Apo-CP向Holo-CP的转化障碍,对于临床难以诊断的WD疑诊患者,采用native-PAGE和Westernblot检测Holo-CP的表达水平是一种较为敏感的诊断指标。
Background
Hepatolenticular degeneration (HLD), also called Wilson disease (WD), is an autosomal recessively recessively inherited disorder of copper metabolism, with an incidence of 15～30 in 1,000,000 in different populations. WD gene, which located in 13q14.3, encoded a 165Kda copper-transporting P-type ATPase (ATP7B) containing 1465 amino acids, and also called ATP7B gene. ATP7B gene mutations can lead to functional deficit of ATP7Base in hepatocytes, which located in trans-Golgi network (TGN) and membrane of bile vessel, and result in transmembrane transport disturbance of copper ion, dyssynthesis of ceruloplasmin (CP) and impaired biliary excretion of copper in hepatocytes. Finally, excess toxic copper accumulate predominantly in the liver, brain, kidney and comea, and caused lower serum CP levels, cirrhosis, neurological/psychiatric symptoms and Kayser-Fleischer ring (K-F ring) in WD patients.
WD is a treatable inherited neurological disease. Most of WD patients can obtain eusemia if they accepted therapy in time. Otherwise, their conditions would be worse or even died. However, many WD patients are often misdiagnosed as other diseases because their early symptoms are complicated and variable and the laboratory copper metabolism tests are often acquired false positive or false negative results in some patients. So it is difficult to make early diagnosis, especially presymptomatic diagnosis and antenatal diagnosis for WD patients before WD gene was cloned.
Many data showed that WD gene mutations were highly genetic heterogeneity. There are more than 300 mutations were found in WD patients and most of them are compound heterozygous mutations. The common mutation types are rare and most of them are scarce mutations. The features of mutations are obviously distinct in different populations. The highly genetic heterogeneity can explain the reasons of complicated and variable symptoms in these patients, but it takes many difficulties for gene diagnosis of WD patients. For this reason, it is a fast and high-performance gene diagnosis strategy for WD patients that identification hot regions of mutations in different ethnic populations, and using techniques such as PCR or multiplex PCR to amplify these regions and scan these regions by sequencing.
Because copper deposition in the liver is usually experienced accumulation stage and saturation stage for WD patients, most of them would be onset between 5 and 35 years. However, recent epidemiological investigations showed that not a few patients present illness after 40 years old. These patients are not typically and can be misdiagnosed as other diseases more often. For WD patients, low serum CP level is a distinctive biochemistry change, and it is the most important evidence for diagnosis too. However, there are 5 percent WD patients with normal CP levels. These 2 special types of WD patients are more often misdiagnosed as other diseases, and gene diagnosis can provide more powerful evidences for them. But few research data was found for these patients.
Sera CP in the body are combined with Holo-CP (conjugated with copper ion) and Apo-CP (not conjugated with copper ion). Recent research showed that transform disturbance from Apo-CP to Holo-CP in WD patients, and their sera Holo-CP expression levels were decreased compared with normal heath person. There have been no report about Holo-CP expression levels in WD patients with normal CP levels from now on.
Based on the progress of gene diagnosis for WD and the foundation of our research on diagnosis and treatment for WD during past decades years, this project plan to find out new hot regions or high frequency spots of gene mutations for Chinese WD patients by restriction mapping analysis and DNA sequencing, and attempt to establish a quick and convenient gene diagnosis method for clinical practice. At the same time, this project plan to collect clinical data and genomic DNA samples of WD patients with late onset or with normal sera CP levels, analyze their clinical features and characters of gene mutations, detect their sera Holo-CP expression levels and attempt to study their molecular pathogenesis.
Objeetives
1. To find out new hot regions or high frequency spots of gene mutations for Chinese WD patients, attempt to establish a quick and convenient gene diagnosis method for clinical practice.
2. To analyze the relationships between the genotypes of high frequency mutation spots of Chinese WD patients and their clinical phenotypes, attempt to study their molecular pathogenesis.
3. To analyze clinical features and characters of gene mutations of Chinese WD patients with late onset or with normal sera CP levels, attempt to study their molecular pathogenesis.
4. To detect sera Holo-CP expression levels of WD patients with normal CP levels, attempt to study their molecular pathogenesis and provide a more sensitive protein marker for them.
Methods
The experiment was composed of four parts.
Chapter 1:Detection for hot regions or high frequency spots of gene mutations of WD.
1. Exon 13 of ATP7B gene in 139 unrelated WD patients which came from middle and eastern China and 52 unrelated normal controls were detected by PCR-DNA sequencing.
2. Exon8,12,13 of ATP7B gene in 142 unrelated WD patients and 52 unrelated normal controls were detected by restriction mapping analysis.
Chapter 2:The relationships between the genotypes of high frequency mutation spots and clinical phenotypes in patients with WD.
The clinical data and results of gene mutations of 142 WD patients which were detected by restriction mapping analysis were collected. Arg778Leu of exon8 and Pro992Leu mutations of exon13 were as the genotypes, and clinical types, gender, age of onset, courses of disease, first symptoms and the detection results of copper metabolism, such as CP and serum copper oxidase (Sco)were as clinical phenotypes. The relationships between the genotypes of high frequency mutation spots of Chinese WD patients and their clinical phenotypes were analyzed.
Chapter 3:Gene diagnosis and the clinical features of WD patients with late-onset and normal sera CP levels.
The clinical data and genomic DNA samples of 13 cases of WD patients with late-onset and 19 cases of WD patients with normal sera CP levels were collected. Their clinical features were analyzed and their mutations were detected by restriction mapping analysis and DNA sequencing including promoter and all exons.
Chapter 4:Detection of sera Holo-CP expression levels of WD patients with normal CP levels and study on their pathogenesis.
Sera samples were collected in 19 WD patients with normal CP levels,30 normal controls and 30 WD patients with lower CP levels. Holo-CP expression levels were detected by native polyacrylamide gel electrophoresis (native-PAGE) and Western blot.
Results
ChapterⅠ:Detection for hot regions or high frequency spots of gene mutations of WD.
1. No abnormality was founded in 52 controls. In 139 patients,6 mutations and 1 polymorphism were found, including 1 novel mutation (Gly988Val). The novel mutation was deposited in GeneBank with the Accession Numbers FJ705811. The compound heterozygous mutation of Arg969Gln/Gly988Val corresponds to severious clinical phenotype. The rate of mutations and polymorphisms of Exon13 was 29.49% (41/139), and its frequency of chromosome mutation was 16.19%(45/278). The rate of Pro992Leu homozygous/heterozygous mutations was 23.02%(32/139), and its frequency of chromosome mutation was 12.59%(35/278).
2. Arg778Leu of exon8 was detected in 48.59%(69/142) patients.13 patients were homozygous mutation and another 56 patients were heterozygous mutation. Thr935Met of exonl2 was detected in 5.63%(8/142) patients and 8 cases were all heterozygous mutation. Pro992Leu of exonl3 was detected in 24.65%(35/142) patients.3 patients were homozygous mutation and 32 patients were heterozygous mutation. Among them,12 cases were compound heterozygous mutation with Arg778Leu/Pro992Leu,1 case was compound heterozygous mutation with Arg778Leu/Thr935Met, and 1 case was Arg778Leu homozygous mutation which combined with Pro992Leu heterozygous mutation. Mutations were detected in 69.01% (98/142) patients totally. The results of this method were confirmed by DNA sequencing.
Chapter 2:The relationships between the genotypes of high frequency mutation spots and clinical phenotypes in patients with WD.
1. There were no relationships between the genotypes of Arg778Leu mutation, Pro992Leu mutation and their clinical phenotypes of clinical types, gender, first symptoms, courses of disease and the levels of CS (P>0.05).
2. The average ages of onset of Arg778Leu homozygous mutation group, Pro992Leu homozygous mutation group and Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than heterozygous mutation group and no Arg778Leu or Pro992Leu mutation group, but there were no statistical significance(P>0.05).
3. The levels of Sco and CP of Arg778Leu homozygous mutation group were lower than no Arg778Leu mutation group significantly (P=0.019, and P=0.011). The levels of Sco and CP of Arg778Leu homozygous mutation group were lower than Arg778Leu heterozygous mutation group, the levels of Sco was no statistical significance(P=0.081) and the levels of CP was statistical significance(P=0.042). The levels of Sco and CP of Arg778Leu heterozygous mutation group were lower than no Arg778Leu mutation group, but no statistical significance(P=0.328, and P= 0.408).
4. The levels of Sco and CP of Pro992Leu homozygous mutation group and Pro992Leu heterozygous mutation group were lower than no Pro992Leu mutation group, and the levels of Sco and CP of Pro992Leu homozygous mutation group were lower than Pro992Leu heterozygous mutation group, but there were all no statistical significance (P>0.05).
5. The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group and Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than no Arg778Leu and no Pro992Leu mutation group significantly (P values were 0.011 and 0.049 for Sco, P values were 0.003 and 0.055 for CP). The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group were lower than only Arg778Leu or Pro992Leu heterozygous group significantly (P value was 0.049 for Sco, P value was 0.009 for CP). The levels of Sco and CP of Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than only Arg778Leu or Pro992Leu heterozygous group, but there were no statistical significance (P>0.05). The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group were lower than Arg778Leu-Pro992Leu compound heterozygous mutation group, but there were no statistical significance (P>0.05).
Chapter 3:Gene diagnosis and the clinical features of WD patients with late-onset and normal sera CP levels.
1. In 13 WD patients with late-onset,6 cases were male and 7 cases were female. Ages of onset were from 40 to 60 years, and their average age of onset was (2.61±2.21) years. Courses of disease were from 0.3 to 8 years and their average course of disease was (2.61±2.21) years.3 cases were patients with hepatic type,7 cases were patients with cerebral type and 3 cases were patients with cerebral-visceral type.6 cases were onset with hepatic symptoms,7 cases were onset with neurological symptoms. Their detection results of Scu, CP and SC were consistent with WD, and 5 of them had abnormal detection results of liver function. They all had comea K-F rings except 1 patient which age of onset was 60 years. The results of abdominal ultrasound examination showed that they all were cirrhosis. Cranium MRI showed abnormal signal in basal ganglia. Compared with patients who were onset with hepatic symptoms, the patients who were onset with neurological symptoms were slow onset and their conditions were less severiouly.
2. There were 6 mutations and 11 polymorphisms in promoter region and all exons of ATP7B gene for these 13 WD patients with late-onset, and 11 cases of them had mutations in exon8,12 and 13. They were all heterozygous mutations or combined heterozygous mutations. The frequency of Arg778Leu was 46.15%(6/13) and Pro992Leu was 30.77%(4/13) in these WD patients. Thr935Met mutation was not found, but 2 cases were detected Arg919Gly heterozygous mutation.
3. In 19 WD patients with normal sera CP levels,9 cases were male and 10 cass were female. Ages of onset were from 4 to 31 years and their average age of onset was (14.68±7.72) years. Courses of disease were from 0.1 to 15 years and their average courses of disease was (3.89±4.09) years.16 cases were patients with hepatic type,1 case was patient with cerebral type,1 case was patient with cerebral-visceral type and 1 case was patient with presymptomatic type.17 cases were onset with hepatic symptoms,2 cases were onset with neurological symptoms. Their detection results of Sco, CP and SC were normal, and 9 of them had abnormal detection results. 12 cases of them had comea K-F rings. The results of abdominal ultrasound examination showed that they all were cirrhosis or acute hepatic injury. Cranium MRI showed abnormal signal in basal ganglia in 2 cases of patients with neurological symptoms.4 cases of them were diagnosed by liver biopsy, and all patients were had final diagnosis by penicillamine challenge test (PCT).
4. There were 11 mutations and 14 polymorphisms in promoter region and all exons of ATP7B gene for these 19 WD patients with normal CP levels.4 mutations including 1870-65G>A, Met645Leu,2575+81A>G and Ala874Pro were novel. In these 19 WD patients,16 cases of them were detected mutations, and any mutations were not found in 3 cases of them. The rate of mutations detection was 84.21% (16/19)。For 16 patients who were detected mutations,3 cases were homozygous mutation,2 cases were compound heterozygous mutation, and only 1 mutation were found in the others cases. The missense mutations is the major mutation types, and 3 kinds of splice junction mutations were found. The frequency of Arg778Leu was 15.79% (3/19), Thr935Met and Pro992Leu mutation were not found.
Chapter 4:Detection of sera Holo-CP expression levels of WD patients with normal CP levels and study on their pathogenesis.
The expression levels of sera Holo-CP of normal controls and WD patients with normal CP levels were higher than WD patients with low CP levels (P=0.000), and the expression levels of sera Holo-CP of WD patients with normal CP levels was lower than normal controls (P=0.013).
Conclusions
1.6 mutations and 1 polymorphism were found in ATP7B gene of 139 WD patients which came from middle and eastern China.
2. Gly988Val, which was a novel mutation, was found in ATP7B gene of 139 WD patients which came from middle and eastern China. Arg969Gln mutation was found first time in Chinese WD patients. The compound heterozygous mutation of Arg969Gln/Gly988Val corresponds to severious clinical phenotype.
3. Exon 13 of ATP7B gene is one of hot mutation regions in Chinese WD patients.
4. The detection rates of gene mutations can be significantly improved by analysis exon8,12 and 13 of WD gene with the method of restriction mapping. The method can help to diagnose suspicious WD cases and their family members in clinical.
5. Pro992Leu mutation is one of high frequency mutation spots in Chinese WD patients.
6. There were no relationships between the genotypes of Arg778Leu mutation, Pro992Leu mutation, Arg778Leu-Pro992Leu compound heterozygous mutation and the clinical phenotypes of clinical types, first symptoms, genders, courses of disease and average ages of onset in 142 Chinese WD patients..
7. The levels of Sco and CP of WD patients with Arg778Leu homozygous mutation, Pro992Leu homozygous mutation, Arg778Leu-Pro992Leu homozygous/ compound heterozygous mutation were lower than those with no Arg778Leu mutation or no Pro992Leu mutation and only Arg778Leu mutation mutation or only Pro992Leu heterozygous mutation.
8. Arg778Leu mutationand Pro992Leu mutation are pathogenic mutations which affected the function of WD protein seriously.
9. Extrapyramidal symptoms were mainly clinical manifestations in 13 WD patients with late-onset which were onset with neurological symptoms. Their liver damage was mild and good effect can be acquired for them by de-copper treatment.
10. Compared with WD patients with late-onset which were onset with neurological symptoms. The patients which were onset with liver symptoms were serious condition. Some of them were accompanied with extrapyramidal symptoms and parts of symptoms can be improved after de-copper treatment.
11.6 mutations and 11 polymorphisms were found in ATP7B gene of 13 WD patients with late-onset.
12. The mutations of 13 WD patients with late-onset were all heterozygous mutations or compound heterozygous mutations, the detection rates of mutations were 46.15% for Arg778Leu heterozygous mutation and 30.77% for Pro992Leu heterozygous mutation in these patients.
13. Liver damage symptoms were mainly clinical manifestations in 19 WD patients with normal CP levels. However, there were patients who were onset with neurological symptoms or without any symptoms.
14.11 mutations and 14 polymorphisms were found in ATP7B gene of 19 WD patients with normal CP levels.
15. The detection rate of Arg778Leu mutation was 15.79% in 19 WD with normal CP levels. Thr935Met mutation and Pro992Leu mutation were not found in these patients. The total detection rate of mutations in exon8,12 and 13 was 42.11%.
16.4 novel mutations were found in ATP7B gene of 19 WD patients with normal CP levels.
17. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in typical WD patients with low CP levels by native-PAGE and Western blot.
18. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in WD patients with mormal CP levels by native-PAGE and Western blot.The detection of Holo-CP expression levels is a sensitive diadynamic criteria for skeptical WD patients in clinical practice.
The originalities of this current project lie in:
1. Gly988Val, which was a novel mutation, was found in ATP7B gene of 139 WD patients which came from middle and eastern China. Arg969Gln mutation was found first time in Chinese WD patients.
2. Exon 13 of ATP7B gene is one of hot mutation regions in Chinese WD patients.
3. Pro992Leu mutation is one of high frequency mutation spots in Chinese WD patients.
4. The levels of Sco and CP of WD patients with Arg778Leu homozygous mutation, Pro992Leu homozygous mutation, Arg778Leu-Pro992Leu homozygous/ compound heterozygous mutation were lower than those with no Arg778Leu mutation or no Pro992Leu mutation and only Arg778Leu mutation mutation or only Pro992Leu heterozygous mutation.
5.6 mutations and 11 polymorphisms were found in ATP7B gene of 13 WD patients with late-onset. These mutations were all heterozygous mutations or compound heterozygous mutations, the detection rates of mutations were 46.15% for Arg778Leu heterozygous mutation and 30.77% for Pro992Leu heterozygous mutation.
6. Liver damage symptoms were mainly clinical manifestations in 19 WD patients with normal CP levels. However, there were patients who were onset with neurological symptoms or without any symptoms.
7.11 mutations and 14 polymorphisms were found in ATP7B gene of 19 WD patients with normal CP levels.4 novel mutations were found in ATP7B gene of these WD patients.
8. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in WD patients with mormal CP levels by native-PAGE and Western blot.The detection of Holo-CP expression levels is a sensitive diadynamic criteria for skeptical WD patients in clinical practice.
引文
[1]Ala A, Walker AP, Ashkan K, et al. Wilson's disease. Lancet,2007;369 (9559): 397-408.
[2]Scheinberg I, Sternlieb I. Wilson's disease. Major Problems in Internal Medicine,1984;23:1-24.
[3]Endo F, Taketa K, Nakamura K, et al. Measurement of blood holoceruloplasmin by EIA using a mouse monoclonal antibody directed to holoceruloplasmin. Implication for mass screening of Wilson disease. Journal of Inherited Metabolic Disease,1994;17(5):616-620.
[4]Ohura T, Abukawa D, Shiraishi H, et al. Pilot study of screening for Wilson disease using dried blood spots obtained from children seen at outpatient clinics. Journal of Inherited Metabolic Disease,1999;22(1):74-80.
[5]Yamaguchi Y, Aoki T, Arashima S, et al. Mass screening for Wilson's disease: results and recommendations. Pediatrics International,1999;41(4):405-408.
[6]Garcia-Villarreal L, Daniels S, Shaw SH, et al. High prevalence of the very rare Wilson disease gene mutation Leu708Pro in the Island of Gran Canaria (Canary Islands, Spain):a genetic and clinical study. Hepatology,2000;32(6): 1329-1336.
[7]Olivarez L, Caggana M, Pass KA, et al. Estimate of the frequency of Wilson's disease in the US Caucasian population:a mutation analysis approach. Annals of Human Genetics,2001;65:459-463.
[8]Hahn SH, Lee SY, Jang YJ, et al. Pilot study of mass screening for Wilson's disease in Korea. Molecular Genetics and Metabolism,2002;76(2):133-136.
[9]Owada M, Suzuki K, Fukushi M, et al. Mass screening for Wilson's disease by measuring urinary holoceruloplasmin. The Journal of Pediatrics,2002;140(5): 614-616.
[10]Dedoussis GV, Genschel J, Sialvera TE, et al. Wilson disease:high prevalence in a mountainous area of Crete. Annals of Human Genetics,2005;69:268-274.
[11]Kroll CA, Ferber MJ, Dawson BD, et al. Retrospective determination of ceruloplasmin in newborn screening blood spots of patients with Wilson disease. Molecular Genetics and Metabolism,2006;89:134-138.
[12]李洵桦,梁秀龄,刘悼霖,等.957例神经遗传病病例分析.中华医学遗传学杂志,1994；11(6)：372-374.
[13]蒋雨平.要提高我国肝豆状核变性病的诊断水平.中华医学杂志,2001；81(11)：641-642.
[14]杨任民.肝豆状核变性.合肥：安徽科技出版社,1995：1-7,28-31,167-205.
[15]Walshe JM. History of Wilson's disease:1912 to 2000. Movement Disorder, 2006;21(2):142-147.
[16]Cumings JN. The copper and iron content of brain and liver in the normal and in hepatolenticular degeneration. Brain,1948;71:410-415.
[17]Scheinberg IH, Gitlin D. Deficiency of caeruloplasmin in patients with hepatolenticular degeneration (Wilson's disease). Science,1952;116:484-485.
[18]Beam AG, Kunkel HG. Biochemical abnormalities in Wilson's disease. The Journal of Clinical Investigation,1952;31:616.
[19]Walshe JM. Penicillamine, a new oral therapy for Wilson's disease. The American Journal of Medicine,1956;21:487-495.
[20]Bull PC, Thomas GR, Rommens JM, et al. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nature Genetics,1993;5:327-337.
[21]Petrukhin K, Fischer SG, Pirastu M, et al. Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nature Genetics,1993;5:338-343.
[22]Tanzi RE, Petrukhin K, Chernov I, et al. The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nature Genetics,1993;5:344-350.
[23]Frydman M, Batsheva BT, Lindsay A, et al. Assignment of the gene for Wilson disease to chromosome 13:Linkage to the Esterase D locus. Proceedings of the National Academy of Sciences,1985;82(3):1819-1821.
[24]Petrukhin K, Lutsenkol S, Chernov I, et al. Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase:genomic organization, alternative splicing, and structure/function predictions. Human Molecular Genetics,1994;3(9):1647-1656.
[25]Fatemi N, Sarkar B. Molecular mechanism of copper transport in Wilson disease. Environmental Health Perspectives,2002;110,695-698.
[26]Lutsenko S, Barnes NL, Bartee MY, et al. Function and regulation of human Copper-Transporting ATPases. Physiological Reviews,2007;87:1011-1046.
[27]Lutsenko S, LeShane ES, Shinde U. Biochemical basis of regμLation of human copper-transporting ATPases. Archives of Biochemistry and Biophysics,2007; 463:134-148.
[28]Fontaine SL, Mercer JFB. Trafficking of the copper-ATPases, ATP7A and ATP7B:Role in copper homeostasis. Archives of Biochemistry and Biophysics, 2007;463:149-167.
[29]Bartee MY, Lutsenko S. Hepatic copper-transporting ATPase ATP7B:function and inactivation at the molecular and cellular level. Biometals,2007;20: 627-637.
[30]Kenney SM, Cox DW. Sequence variation database for the Wilson Disease copper transporter, ATP7B. Human Mutation,2007;28(12):1171-1177.
[31]胡纪源,吕达平,王共强,等.肝豆状核变性的临床误诊研究.中华医学杂志,2001：81：642-644.
[32]Roberts EA, Schilsky ML. A Practice Guideline on Wilson Disease. Hepatology, 2003;37(6):1475-1492.
[33]Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease:An update. Hepatology,2008;47(6):2089-2111.
[34]Figus A, Iampis R, Devoto M, et al.Carrier detection and early diagnosis of Wilson disease by restriction fragment length polymorphism analysis. Journal of Medical Genetics,1989;26(2):78-82.
[35]吴志英,王柠,慕容慎行,余龙.WD基因内部及其两侧翼的微卫星多态快速检出Wilson's病基因携带者及症状前患者.中国神经精神疾病杂志,1996；22(1)：61-63.
[36]王丽娟,梁秀龄,刘焯霖,等.中国南方汉人D13S316位点多态性及Wilson病基因诊断的研究.中国神经精神疾病杂志,2000；26(2)：90-92.
[37]Gupta A, Maulik M, Nasipuri P, et al. Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers. Clinical Chemistry,2007;53(9):1601-1608.
[38]Harmut H, Schmidt J. Introducing single-nucleotide polymorphism markers in the diagnosis of Wilson disease. Clinical Chemistry,2007;53(9):1568-1569.
[39]Thomas GR, Forbes J R, Roberts EA, et al. The Wilson disease gene:Spectrum of mutations and their consequences. Nature Genetics,1995;9(2):210-217.
[40]Shah AB, Chernov I, Zhang HT, et al. Identification and analysis of mutations in the Wilson disease gene (ATP7B):population frequencies, genotype-phenotype correlation, and functional analyses. American journal of human genetics,1997;61:317-328.
[41]Duc HH, Hefter H, Stremmel W, et al. His1069Gln and six novel Wilson disease mutations:analysis of relevance for early diagnosis and phenotype. European Journal of Human Genetics,1998;6:616-623.
[42]Curtis D, Durkie M, Balac P, et al. A study of Wilson disease mutations in Britain. Human mutation,1999;14:304-311.
[43]Firneisz G, Lakatos PL, Szalay F, et al. Common mutation of ATP7B in Wilson disease patients from Hungary. American Journal of Medical Genetics,2002,15; 108(1):23-28.
[44]杨任民,范玉新,余龙,等.肝豆状核变性基因的一种新型错义突变.中华医学杂志,1997；77(5)：344-347.
[45]吴志英,慕容慎行,王柠,等.中国人肝豆状核变性基因第14、18和5号外显子突变及多态研究.临床神经病学杂志,1997；10(5)：259-261.
[46]马少春,徐评议,梁秀龄,等.肝豆状核变性8号14号外显子基因突变的检测.中山医科大学学报,1998；19(1)：14-17,26.
[47]Vrabelova S, Letocha O, Borsky M, et al. Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease. Molecular Genetics and Metabolism,2005;86:277-285.
[48]杨静芳,梁秀龄,马守忠,等。74例肝豆状核变性患者中ATP7B基因七种新突变的发现.中华神经科杂志,2006；39(10)：673-677.
[49]Waldenstrom, E, Lagerkvist A, Dahlman T, et al. Efficient detection of mutations in Wilson disease by manifold sequencing. Genomics,1996;37: 303-309.
[50]许月芳,范玉新,余龙,等.PCR直接测序在Wilson病基因第8外显子检出一个突变热点.中华医学遗传学杂志,1998；15(5)：284-287.
[51]徐评议,梁秀龄,马少春.Wilson's病8号外显子突变研究.中华医学遗传学杂志,1999；16(2)：88-89.
[52]吴志英,王柠,慕容慎行,等.肝豆状核变性基因第12号外显子突变特征的研究.中华医学杂志,1999；79(6)：422-424.
[53]吴志英,王柠,林珉婷,等.Wilson病基因全长外显子的突变检测和分析.中华神经科杂志,2001；34(3)：152-155.
[54]Lipshutz R, Fodor S, Gingeras T, et al. High density synthetic oligonucleotide arrays. Nature Genetics Supplement,1999;21:20-24.
[55]Baner J, Isaksson A, Waldenstrom E, et al. Parallel gene analysis with allele-specific padlock probes and tag microarrays. Nucleic Acids Research, 2003; 31(17):103-109.
[56]http://www.wilsondisease.med.ualberta.ca/database.asp
[57]Ferenci P. Regional distribution of mutations of the ATP7B gene in patients with Wilson disease:impact on genetic testing. Human Genetics,2006,120(2): 151-159.
[58]Maier DT, Ferenci P, Polli C, et al. Detection of the His1069Glu mutation in Wilson disease by rapid polymerase chain reaction. Annals of Internal Medicine, 1997;127(1):70-72.
[59]马少春,徐评议,梁秀龄,等.肝豆状核变性8号外显子778密码子基因突变的酶切检测.中国神经精神疾病杂志,1998；24(6)：333-335.
[60]王柠,吴志英,林珉婷,等.应用限制性酶谱分析法快速检出Wilson病基因突变热点.中华神经科杂志,1999；32(5)：281-283.
[61]Reddy K. Late onset Wilson disease frequently overlooked. Gastroenterology, 2006; 131:343.
[62]Ferenci P, Czlonkowska A, Merle U, et al. Late-onset Wilson's disease. Gastroenterology,2007;132:1294-1298.
[63]Sohtaoglu M, Ergin H, Ozekmekci S, et al. Patient with late-onset Wilson's disease:deterioration with penicillamine. Movement Disorder,2007;22: 290-291.
[64]Ala A, Borjigin J, Rochwarger A, et al. Wilson disease in septuagenarian siblings:raising the bar for diagnosis. Hepatology,2005;41:668-670.
[65]Walshe JM, Waldenstrom E, Sams V, et al. Abdominal malignancies in patients with Wilson's disease. Quarterly Journal of Medicine,2003;96:657-662.
[66]Czlonkowska A, Rodo M, Gromadzka G. Late Onset Wilson's disease: Therapeutic implications. Movement Disorders,2008;23(6):896-898.
[67]Hirano K, Ogihara T, Ogihara H, et al. Identification of apo- and holo-forms of ceruloplasmin in patients with Wilson's disease using native polyacrylamide gel electrophoresis. Clinical Biochemistry,2005;38:9-12.
[68]中华医学会神经病学分会帕金森病及运动障碍学组,中华医学会神经病学分会神经遗传病学组.肝豆状核变性的诊断与治疗指南.中华神经科杂志,2008；41(8)：566-569.
[69]Nanji MS, Nguyen VT, Kawasoe JH, et al. Haplotype and mutation analysis in Japanese patients with Wilson disease. The American Journal of Human Genetics,1997; 60:1423-1429.
[70]Tsai CH, Tsai FJ, Wu JY, et al. Mutation analysis of Wilson disease in Taiwan and description of six new mutations. Human mutation,1998,12:370-376.
[71]Gu YH, Kodama H, Du S-L, et al. Mutation spectrum and polymorphisms in ATP7B identified on direct sequencing of all exons in Chinese Han and Hui ethnicpatients with Wilson's disease. Clinical Genetics,2003;64:479-484.
[72]Chloe MM, Ching WL, Sidney T, et al. Mutational analysis of 65 Wilson disease patients in Hong Kong Chinese:Identification of 17 novel mutations and its genetic heterogeneity. Journal of Human Genetics,2008;53:55-63.
[73]程楠,洪铭范,胡纪源,等.应用双重PCR-SSCP检测Wilson病患者的基因突变.医学研究杂志,2008；37：40-42.
[74]http://www.faqs.org/health/bios/5/Werner-Arber.html
[75]杨任民,杨兴涛,鲍远程,等.肝豆状核变性症临床分型的初步探讨.天津医学,1983；5：288-291.
[76]Machado AA, Deguti MM, Genschel J, et al. Neurological manifestations and ATP7B mutations in Wilson's disease. Parkinsonism Related Disorder,2008; 14(3):246-249.
[77]黄帆,梁秀龄,王莹,等.肝豆状核变性基因Arg778Leu突变与临床表型 关系研究.中华神经科杂志,2000；33(5)：290-291.
[78]刘晓青,张建明,鲍克容,等.Wilson病ATP7B基因Arg778Leu突变与临床表型之相关性探讨.中华儿科杂志,1999,37(6)：359-361.
[79]吴志英,王柠,林珉婷,等.Wilson病基因突变类型与临床表型的关系.中华医学杂志,2003；83(4)：309-311.
[80]Terada K, Schilskyb M, Miura N, et al. ATP7B (WND) protein. The International Journal of Biochemistry & Cell Biology,1998,30:1063-1067.
[81]Forbes JR, Cox DW. Functional characterization of missense mutations in ATP7B:Wilson disease mutation or normal variant? American journal of human genetics,1998;63:1663-1674.
[82]Schaeffer M, Hopkins RG, Failla ML, et al. Hepatocyte-specific localization and copper-dependent trafficking of the Wilson's disease protein in the liver. American Journal of Physiology-Gastrointestinal and Liver Physiology,1999; 276:639-646.
[83]Forbes JR, Cox DW. Copper-independet trafficking of Wilson disease mutant ATP7B proteins. Human Molecular Genetics,2000;9(13):1927-1935.
[84]Chappuisa P, Calleberta J, Quignona V et al. Late neurological presentations of Wilson disease patients in French population and identification of 8 novel mutations in the ATP7B gene. Journal of Trace Elements in Medicine and Biology,2007;21:37-42.
[85]杨任民,鲍远程,杨兴涛,等.晚发性肝豆状核变性.中华神经精神科杂志,1985；18：238.
[86]张永红,杨旭,唐晓鹏,等.迟发型肝豆状核变性的临床特点和治疗转归.中南大学学报(医学版),2009；34(1)：40-44.
[87]Cauza E, Maier-Dobersberger T, Polli C, et al. Screening for Wilson's disease in patients with liver diseases by serum ceruloplasmin. Journal of Hepatology, 1997,27:358-362.
[88]Steindl P, Ferenci P, Dienes HP, Madl C, et al. Wilson's disease in patients presenting with liver disease:a diagnostic challenge. Gastroenterology,1997; 113:212-218.
[89]Gow PJ, Smallwood RA, Angus PW, et al. Diagnosis of Wilson's disease:an experience over three decades. Gut,2000; 46:415-419.
[90]Weiss KH, Merle U, Schaefer M, et al. Copper toxicosis gene MURR1 is not changed in Wilson disease patients with normal blood ceruloplasmin levels. World Journal of Gastroenterology,2006;12(14):2239-2242.
[91]Sato M, Gitlin JD. Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin. The Journal of Biological Chemistry,1991;266(6): 5128-5134.
[92]Harris ZL, Gitlin JD. Genetic and molecular basis for copper toxicity. The American Journal of Clinical Nutrition,1996;63:836-841.
[93]Hellman NE, Kono S, Mancini GM, et al. Mechanisms of copper incorporation into human ceruloplasmin. The Journal of Biological Chemistry,2002;277: 46632-46638.
[94]Terada K, Kawarada Y, Miura N, et al. Copper incorporation into ceruloplasmin in rat livers. Biochim Biophys Acta,1995;1270:58-62.
[95]Funmei Y, Susan L, Naylor, et al. Characterization, mapping, and expression of the human ceruloplasmin gene. Proceedings of the National Academy of Sciences,1986;83:3257-3261.
[96]Edwards CQ, Williams DM, Cartwright GE. Hereditary hypoceruloplasminemia. Clinical Genetics,1979;15:311-316.
[97]Harris, ZL, Klomp LWJ, Gitlin JD. Aceruloplasminemia:an inherited neurodegenerative disease with impairment of iron homeostasis. The American Journal of Clinical Nutrition,1998;67:972-977.
[98]Sedlak E, Pernilla WS. Discrete roles of copper ions in chemical unfolding of human ceruloplasmin. Biochemistry,2007;46 (33):9638-9644.
[99]van de Sluis BJ, Breen M, Nanji M, et al. Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16. Human Molecular Genetics, 1999; 8:501-507.
[100]Tao TY, Liu F, Klomp L, Wijmenga C, Gitlin JD. The copper toxicosis gene product Murrl directly interacts with the Wilson disease protein. The Journal of Biological Chemistry,2003;278:41593-41596.
[101]Zhi-Ying Wu, Gui-Xian Zhao, Wan-Jin Chen, et al. Mutation analysis of 218 Chinese patients with Wilson disease revealed no correlation between the canine copper toxicosis gene MURR1 and Wilson disease. The Journal of Molecular Medicine,2006;84:438-442.
[102]Gibbs K, Walshe JM. Biliary excretion of copper in Wilson's disease. Lancet, 1980; 2:538.
[103]Lyengar V, Brewer GJ, Dick RD, et al. Studies of the cholecy stokinin-stimulated biliary secretions reveal a high molecular weight copper-binding substance in normal subjects that is absent in patients with wilson's disease. The Journal of Laboratory and Clinical Medicine,1988;111: 267.
[104]Ren MS, Fan YX, Yang RM, et al. A comparative study of biliary elements and clinical phenotypes in Wilson disease. China National Journal of New Gastroenterology,1997;3(4):260-262.
[105]胡文彬,杨任民.肝豆片Ⅰ号对肝豆状核变性患者胆汁微量元素影响的前瞻性研究.中国中西医结合杂志,2001；21(7)：507-509.
[1]Walshe JM. History of Wilson's Disease:1912 to 2000. Movement Disorders, 2006,21(2):142-147.
[2]Ala A, Walker AP, Ashkan K, et al. Wilson's disease. Lancet,2007,369: 397-408.
[3]胡纪源,吕达平,王共强,等.肝豆状核变性的临床误诊研究.中华医学杂志,2001,81(11)：642-644.
[4]Roberts EA, Schilsky ML. A Practice Guideline on Wilson Disease. Hepatology, 2003,37(6):1475-1492.
[5]Bull PC, Thomas GR, Rommens JM, et al. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nature Genetics,1993,5:327-337.
[6]Petrukhin K, Fischer SG, Pirastu M, et al. Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nature Genetics,1993,5:338-343.
[7]Tanzi RE, Petrukhin K, Chernov I, et al. The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nature Genetics,1993,5:344-350.
[8]Ferenci P. Regional distribution of mutations of the ATP7B gene in patients with Wilson disease:impact on genetic testing. Human Genetics,2006,120: 151-159.
[9]Figus A, Lampis R, Devoto M, et al. Carrier detection and early diagnosis of Wilson's disease by restriction fragment length polymorphism analysis. Journal of Medical Genetics,1989,26(2):78-82.
[10]吴志英,王柠,慕容慎行,等.WD基因内部及其两侧翼的微卫星多态快速检出Wilson's病基因携带者及症状前患者.中国神经精神疾病杂志,1996,22(1)：1-3,18,62.
[11]王丽娟,梁秀龄,刘焯霖,等.中国南方汉人D13S316位点多态性及Wilson病基因诊断的研究.中国神经精神疾病杂志,2000,26(2)：90-92.
[12]Gupta A, Maulik M, Nasipuri P, et al. Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers. Clinical Chemistry,2007,53(9):1601-1608.
[13]Harmut H, Schmidt J. Introducing single-nucleotide polymorphism markers in the diagnosis of Wilson disease. Clinical Chemistry,2007,53(9):1568-1569.
[14]Fujita K, Silver J. Single-strand conformational polymorphism. Genome Research,1994,4:137-140.
[15]Thomas GR, Forbes JR, Roberts EA, et al. The Wilson disease gene:spectrum of mutation and their consequences. Nature Genetics,1995;9:210-217.
[16]杨任民,范玉新,余龙,等.肝豆状核变性基因的一种新型错义突变.中华医学杂志,1997；77(5)：344-347.
[17]吴志英,慕容慎行,王柠,等。中国人肝豆状核变性基因第14、18和5号外显子突变及多态研究.临床神经病学杂志,1997,10(5)：259-261.
[18]马少春,徐评议,梁秀龄,等.肝豆状核变性8号14号外显子基因突变的检测.中山医科大学学报,1998,19(1)：14-17,26.
[19]程楠,洪铭范,胡纪源,等.应用双重PCR-SSCP检测Wilson病患者的基因突变.医学研究杂志,2008,37(10)：40-42.
[20]Maier DT, Ferenci P, Polli C, et al. Detection of the His1069Glu mutation in Wilson disease by rapid polymerase chain reaction. Annals of Internal Medicine, 1997,127(1):21-26.
[21]马少春,徐评议,梁秀龄,等.肝豆状核变性8号外显子778密码子基因突变的酶切检测.中国神经精神疾病杂志,1998,24(6)：333-335,381.
[22]王柠,吴志英,林珉婷,等.应用限制性酶谱分析法快速检出Wilson病基因突变热点.中华神经科杂志,1999,32(5)：281-283.
[23]程楠,杨任民,洪铭范,等.PCR-酶切法检测Wilson病935密码子基因突变.中国优生与遗传杂志,2000,8(3)：24-25.
[24]黄帆,梁秀龄,徐评议,等.用荧光PCR对中国人肝豆状核变性进行早期诊断及携带者检测.中华医学遗传学杂志,2001,18(1)：17-20.
[25]Sivakumaran TA, Kucheria K, Oefner PJ. Denaturing high performance liquid chromatography in the molecular diagnosis of genetic disorders. Current Science,2003,84(3):291-296.
[26]Vrabelova S, Letocha O, Borsky M, et al. Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease. Molecular Genetics and Metabolism,2005,86:277-285.
[27]杨静芳,梁秀龄,马守忠,等。74例肝豆状核变性患者中ATP7B基因七种新突变的发现.中华神经科杂志,2006,39(10)：673-677.
[28]Waldenstrom E, Lagerkvist A, Dahlman T, et al. Efficient detection of mutations in Wilson disease by manifold sequencing. Genomics,1996,37: 303-309.
[29]吴志英,王柠,林珉婷,等.Wilson病基因全长外显子的突变检测和分析.中华神经科杂志,2001,34(3)：152-155.
[30]Gresham D, Dunham MJ, Botstein D. Comparing whole genomes using DNA microarrays. Nature Reviews Genetics,2008,9:291-302.
[31]Baner J, Isaksson A, Waldenstrom E, et al. Parallel gene analysis with allele-specific padlock probes and tag microarrays. Nucleic Acids Research, 2003,31(17):103-109.
[2]Scheinberg I, Sternlieb I. Wilson's disease. Major Problems in Internal Medicine,1984;23:1-24.
[3]Endo F, Taketa K, Nakamura K, et al. Measurement of blood holoceruloplasmin by EIA using a mouse monoclonal antibody directed to holoceruloplasmin. Implication for mass screening of Wilson disease. Journal of Inherited Metabolic Disease,1994;17(5):616-620.
[4]Ohura T, Abukawa D, Shiraishi H, et al. Pilot study of screening for Wilson disease using dried blood spots obtained from children seen at outpatient clinics. Journal of Inherited Metabolic Disease,1999;22(1):74-80.
[5]Yamaguchi Y, Aoki T, Arashima S, et al. Mass screening for Wilson's disease: results and recommendations. Pediatrics International,1999;41(4):405-408.
[6]Garcia-Villarreal L, Daniels S, Shaw SH, et al. High prevalence of the very rare Wilson disease gene mutation Leu708Pro in the Island of Gran Canaria (Canary Islands, Spain):a genetic and clinical study. Hepatology,2000;32(6): 1329-1336.
[7]Olivarez L, Caggana M, Pass KA, et al. Estimate of the frequency of Wilson's disease in the US Caucasian population:a mutation analysis approach. Annals of Human Genetics,2001;65:459-463.
[8]Hahn SH, Lee SY, Jang YJ, et al. Pilot study of mass screening for Wilson's disease in Korea. Molecular Genetics and Metabolism,2002;76(2):133-136.
[9]Owada M, Suzuki K, Fukushi M, et al. Mass screening for Wilson's disease by measuring urinary holoceruloplasmin. The Journal of Pediatrics,2002;140(5): 614-616.
[10]Dedoussis GV, Genschel J, Sialvera TE, et al. Wilson disease:high prevalence in a mountainous area of Crete. Annals of Human Genetics,2005;69:268-274.
[11]Kroll CA, Ferber MJ, Dawson BD, et al. Retrospective determination of ceruloplasmin in newborn screening blood spots of patients with Wilson disease. Molecular Genetics and Metabolism,2006;89:134-138.
[12]李洵桦,梁秀龄,刘悼霖,等.957例神经遗传病病例分析.中华医学遗传学杂志,1994；11(6)：372-374.
[13]蒋雨平.要提高我国肝豆状核变性病的诊断水平.中华医学杂志,2001；81(11)：641-642.
[14]杨任民.肝豆状核变性.合肥：安徽科技出版社,1995：1-7,28-31,167-205.
[15]Walshe JM. History of Wilson's disease:1912 to 2000. Movement Disorder, 2006;21(2):142-147.
[16]Cumings JN. The copper and iron content of brain and liver in the normal and in hepatolenticular degeneration. Brain,1948;71:410-415.
[17]Scheinberg IH, Gitlin D. Deficiency of caeruloplasmin in patients with hepatolenticular degeneration (Wilson's disease). Science,1952;116:484-485.
[18]Beam AG, Kunkel HG. Biochemical abnormalities in Wilson's disease. The Journal of Clinical Investigation,1952;31:616.
[19]Walshe JM. Penicillamine, a new oral therapy for Wilson's disease. The American Journal of Medicine,1956;21:487-495.
[20]Bull PC, Thomas GR, Rommens JM, et al. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nature Genetics,1993;5:327-337.
[21]Petrukhin K, Fischer SG, Pirastu M, et al. Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nature Genetics,1993;5:338-343.
[22]Tanzi RE, Petrukhin K, Chernov I, et al. The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nature Genetics,1993;5:344-350.
[23]Frydman M, Batsheva BT, Lindsay A, et al. Assignment of the gene for Wilson disease to chromosome 13:Linkage to the Esterase D locus. Proceedings of the National Academy of Sciences,1985;82(3):1819-1821.
[24]Petrukhin K, Lutsenkol S, Chernov I, et al. Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase:genomic organization, alternative splicing, and structure/function predictions. Human Molecular Genetics,1994;3(9):1647-1656.
[25]Fatemi N, Sarkar B. Molecular mechanism of copper transport in Wilson disease. Environmental Health Perspectives,2002;110,695-698.
[26]Lutsenko S, Barnes NL, Bartee MY, et al. Function and regulation of human Copper-Transporting ATPases. Physiological Reviews,2007;87:1011-1046.
[27]Lutsenko S, LeShane ES, Shinde U. Biochemical basis of regμLation of human copper-transporting ATPases. Archives of Biochemistry and Biophysics,2007; 463:134-148.
[28]Fontaine SL, Mercer JFB. Trafficking of the copper-ATPases, ATP7A and ATP7B:Role in copper homeostasis. Archives of Biochemistry and Biophysics, 2007;463:149-167.
[29]Bartee MY, Lutsenko S. Hepatic copper-transporting ATPase ATP7B:function and inactivation at the molecular and cellular level. Biometals,2007;20: 627-637.
[30]Kenney SM, Cox DW. Sequence variation database for the Wilson Disease copper transporter, ATP7B. Human Mutation,2007;28(12):1171-1177.
[31]胡纪源,吕达平,王共强,等.肝豆状核变性的临床误诊研究.中华医学杂志,2001：81：642-644.
[32]Roberts EA, Schilsky ML. A Practice Guideline on Wilson Disease. Hepatology, 2003;37(6):1475-1492.
[33]Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease:An update. Hepatology,2008;47(6):2089-2111.
[34]Figus A, Iampis R, Devoto M, et al.Carrier detection and early diagnosis of Wilson disease by restriction fragment length polymorphism analysis. Journal of Medical Genetics,1989;26(2):78-82.
[35]吴志英,王柠,慕容慎行,余龙.WD基因内部及其两侧翼的微卫星多态快速检出Wilson's病基因携带者及症状前患者.中国神经精神疾病杂志,1996；22(1)：61-63.
[36]王丽娟,梁秀龄,刘焯霖,等.中国南方汉人D13S316位点多态性及Wilson病基因诊断的研究.中国神经精神疾病杂志,2000；26(2)：90-92.
[37]Gupta A, Maulik M, Nasipuri P, et al. Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers. Clinical Chemistry,2007;53(9):1601-1608.
[38]Harmut H, Schmidt J. Introducing single-nucleotide polymorphism markers in the diagnosis of Wilson disease. Clinical Chemistry,2007;53(9):1568-1569.
[39]Thomas GR, Forbes J R, Roberts EA, et al. The Wilson disease gene:Spectrum of mutations and their consequences. Nature Genetics,1995;9(2):210-217.
[40]Shah AB, Chernov I, Zhang HT, et al. Identification and analysis of mutations in the Wilson disease gene (ATP7B):population frequencies, genotype-phenotype correlation, and functional analyses. American journal of human genetics,1997;61:317-328.
[41]Duc HH, Hefter H, Stremmel W, et al. His1069Gln and six novel Wilson disease mutations:analysis of relevance for early diagnosis and phenotype. European Journal of Human Genetics,1998;6:616-623.
[42]Curtis D, Durkie M, Balac P, et al. A study of Wilson disease mutations in Britain. Human mutation,1999;14:304-311.
[43]Firneisz G, Lakatos PL, Szalay F, et al. Common mutation of ATP7B in Wilson disease patients from Hungary. American Journal of Medical Genetics,2002,15; 108(1):23-28.
[44]杨任民,范玉新,余龙,等.肝豆状核变性基因的一种新型错义突变.中华医学杂志,1997；77(5)：344-347.
[45]吴志英,慕容慎行,王柠,等.中国人肝豆状核变性基因第14、18和5号外显子突变及多态研究.临床神经病学杂志,1997；10(5)：259-261.
[46]马少春,徐评议,梁秀龄,等.肝豆状核变性8号14号外显子基因突变的检测.中山医科大学学报,1998；19(1)：14-17,26.
[47]Vrabelova S, Letocha O, Borsky M, et al. Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease. Molecular Genetics and Metabolism,2005;86:277-285.
[48]杨静芳,梁秀龄,马守忠,等。74例肝豆状核变性患者中ATP7B基因七种新突变的发现.中华神经科杂志,2006；39(10)：673-677.
[49]Waldenstrom, E, Lagerkvist A, Dahlman T, et al. Efficient detection of mutations in Wilson disease by manifold sequencing. Genomics,1996;37: 303-309.
[50]许月芳,范玉新,余龙,等.PCR直接测序在Wilson病基因第8外显子检出一个突变热点.中华医学遗传学杂志,1998；15(5)：284-287.
[51]徐评议,梁秀龄,马少春.Wilson's病8号外显子突变研究.中华医学遗传学杂志,1999；16(2)：88-89.
[52]吴志英,王柠,慕容慎行,等.肝豆状核变性基因第12号外显子突变特征的研究.中华医学杂志,1999；79(6)：422-424.
[53]吴志英,王柠,林珉婷,等.Wilson病基因全长外显子的突变检测和分析.中华神经科杂志,2001；34(3)：152-155.
[54]Lipshutz R, Fodor S, Gingeras T, et al. High density synthetic oligonucleotide arrays. Nature Genetics Supplement,1999;21:20-24.
[55]Baner J, Isaksson A, Waldenstrom E, et al. Parallel gene analysis with allele-specific padlock probes and tag microarrays. Nucleic Acids Research, 2003; 31(17):103-109.
[56]http://www.wilsondisease.med.ualberta.ca/database.asp
[57]Ferenci P. Regional distribution of mutations of the ATP7B gene in patients with Wilson disease:impact on genetic testing. Human Genetics,2006,120(2): 151-159.
[58]Maier DT, Ferenci P, Polli C, et al. Detection of the His1069Glu mutation in Wilson disease by rapid polymerase chain reaction. Annals of Internal Medicine, 1997;127(1):70-72.
[59]马少春,徐评议,梁秀龄,等.肝豆状核变性8号外显子778密码子基因突变的酶切检测.中国神经精神疾病杂志,1998；24(6)：333-335.
[60]王柠,吴志英,林珉婷,等.应用限制性酶谱分析法快速检出Wilson病基因突变热点.中华神经科杂志,1999；32(5)：281-283.
[61]Reddy K. Late onset Wilson disease frequently overlooked. Gastroenterology, 2006; 131:343.
[62]Ferenci P, Czlonkowska A, Merle U, et al. Late-onset Wilson's disease. Gastroenterology,2007;132:1294-1298.
[63]Sohtaoglu M, Ergin H, Ozekmekci S, et al. Patient with late-onset Wilson's disease:deterioration with penicillamine. Movement Disorder,2007;22: 290-291.
[64]Ala A, Borjigin J, Rochwarger A, et al. Wilson disease in septuagenarian siblings:raising the bar for diagnosis. Hepatology,2005;41:668-670.
[65]Walshe JM, Waldenstrom E, Sams V, et al. Abdominal malignancies in patients with Wilson's disease. Quarterly Journal of Medicine,2003;96:657-662.
[66]Czlonkowska A, Rodo M, Gromadzka G. Late Onset Wilson's disease: Therapeutic implications. Movement Disorders,2008;23(6):896-898.
[67]Hirano K, Ogihara T, Ogihara H, et al. Identification of apo- and holo-forms of ceruloplasmin in patients with Wilson's disease using native polyacrylamide gel electrophoresis. Clinical Biochemistry,2005;38:9-12.
[68]中华医学会神经病学分会帕金森病及运动障碍学组,中华医学会神经病学分会神经遗传病学组.肝豆状核变性的诊断与治疗指南.中华神经科杂志,2008；41(8)：566-569.
[69]Nanji MS, Nguyen VT, Kawasoe JH, et al. Haplotype and mutation analysis in Japanese patients with Wilson disease. The American Journal of Human Genetics,1997; 60:1423-1429.
[70]Tsai CH, Tsai FJ, Wu JY, et al. Mutation analysis of Wilson disease in Taiwan and description of six new mutations. Human mutation,1998,12:370-376.
[71]Gu YH, Kodama H, Du S-L, et al. Mutation spectrum and polymorphisms in ATP7B identified on direct sequencing of all exons in Chinese Han and Hui ethnicpatients with Wilson's disease. Clinical Genetics,2003;64:479-484.
[72]Chloe MM, Ching WL, Sidney T, et al. Mutational analysis of 65 Wilson disease patients in Hong Kong Chinese:Identification of 17 novel mutations and its genetic heterogeneity. Journal of Human Genetics,2008;53:55-63.
[73]程楠,洪铭范,胡纪源,等.应用双重PCR-SSCP检测Wilson病患者的基因突变.医学研究杂志,2008；37：40-42.
[74]http://www.faqs.org/health/bios/5/Werner-Arber.html
[75]杨任民,杨兴涛,鲍远程,等.肝豆状核变性症临床分型的初步探讨.天津医学,1983；5：288-291.
[76]Machado AA, Deguti MM, Genschel J, et al. Neurological manifestations and ATP7B mutations in Wilson's disease. Parkinsonism Related Disorder,2008; 14(3):246-249.
[77]黄帆,梁秀龄,王莹,等.肝豆状核变性基因Arg778Leu突变与临床表型 关系研究.中华神经科杂志,2000；33(5)：290-291.
[78]刘晓青,张建明,鲍克容,等.Wilson病ATP7B基因Arg778Leu突变与临床表型之相关性探讨.中华儿科杂志,1999,37(6)：359-361.
[79]吴志英,王柠,林珉婷,等.Wilson病基因突变类型与临床表型的关系.中华医学杂志,2003；83(4)：309-311.
[80]Terada K, Schilskyb M, Miura N, et al. ATP7B (WND) protein. The International Journal of Biochemistry & Cell Biology,1998,30:1063-1067.
[81]Forbes JR, Cox DW. Functional characterization of missense mutations in ATP7B:Wilson disease mutation or normal variant? American journal of human genetics,1998;63:1663-1674.
[82]Schaeffer M, Hopkins RG, Failla ML, et al. Hepatocyte-specific localization and copper-dependent trafficking of the Wilson's disease protein in the liver. American Journal of Physiology-Gastrointestinal and Liver Physiology,1999; 276:639-646.
[83]Forbes JR, Cox DW. Copper-independet trafficking of Wilson disease mutant ATP7B proteins. Human Molecular Genetics,2000;9(13):1927-1935.
[84]Chappuisa P, Calleberta J, Quignona V et al. Late neurological presentations of Wilson disease patients in French population and identification of 8 novel mutations in the ATP7B gene. Journal of Trace Elements in Medicine and Biology,2007;21:37-42.
[85]杨任民,鲍远程,杨兴涛,等.晚发性肝豆状核变性.中华神经精神科杂志,1985；18：238.
[86]张永红,杨旭,唐晓鹏,等.迟发型肝豆状核变性的临床特点和治疗转归.中南大学学报(医学版),2009；34(1)：40-44.
[87]Cauza E, Maier-Dobersberger T, Polli C, et al. Screening for Wilson's disease in patients with liver diseases by serum ceruloplasmin. Journal of Hepatology, 1997,27:358-362.
[88]Steindl P, Ferenci P, Dienes HP, Madl C, et al. Wilson's disease in patients presenting with liver disease:a diagnostic challenge. Gastroenterology,1997; 113:212-218.
[89]Gow PJ, Smallwood RA, Angus PW, et al. Diagnosis of Wilson's disease:an experience over three decades. Gut,2000; 46:415-419.
[90]Weiss KH, Merle U, Schaefer M, et al. Copper toxicosis gene MURR1 is not changed in Wilson disease patients with normal blood ceruloplasmin levels. World Journal of Gastroenterology,2006;12(14):2239-2242.
[91]Sato M, Gitlin JD. Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin. The Journal of Biological Chemistry,1991;266(6): 5128-5134.
[92]Harris ZL, Gitlin JD. Genetic and molecular basis for copper toxicity. The American Journal of Clinical Nutrition,1996;63:836-841.
[93]Hellman NE, Kono S, Mancini GM, et al. Mechanisms of copper incorporation into human ceruloplasmin. The Journal of Biological Chemistry,2002;277: 46632-46638.
[94]Terada K, Kawarada Y, Miura N, et al. Copper incorporation into ceruloplasmin in rat livers. Biochim Biophys Acta,1995;1270:58-62.
[95]Funmei Y, Susan L, Naylor, et al. Characterization, mapping, and expression of the human ceruloplasmin gene. Proceedings of the National Academy of Sciences,1986;83:3257-3261.
[96]Edwards CQ, Williams DM, Cartwright GE. Hereditary hypoceruloplasminemia. Clinical Genetics,1979;15:311-316.
[97]Harris, ZL, Klomp LWJ, Gitlin JD. Aceruloplasminemia:an inherited neurodegenerative disease with impairment of iron homeostasis. The American Journal of Clinical Nutrition,1998;67:972-977.
[98]Sedlak E, Pernilla WS. Discrete roles of copper ions in chemical unfolding of human ceruloplasmin. Biochemistry,2007;46 (33):9638-9644.
[99]van de Sluis BJ, Breen M, Nanji M, et al. Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16. Human Molecular Genetics, 1999; 8:501-507.
[100]Tao TY, Liu F, Klomp L, Wijmenga C, Gitlin JD. The copper toxicosis gene product Murrl directly interacts with the Wilson disease protein. The Journal of Biological Chemistry,2003;278:41593-41596.
[101]Zhi-Ying Wu, Gui-Xian Zhao, Wan-Jin Chen, et al. Mutation analysis of 218 Chinese patients with Wilson disease revealed no correlation between the canine copper toxicosis gene MURR1 and Wilson disease. The Journal of Molecular Medicine,2006;84:438-442.
[102]Gibbs K, Walshe JM. Biliary excretion of copper in Wilson's disease. Lancet, 1980; 2:538.
[103]Lyengar V, Brewer GJ, Dick RD, et al. Studies of the cholecy stokinin-stimulated biliary secretions reveal a high molecular weight copper-binding substance in normal subjects that is absent in patients with wilson's disease. The Journal of Laboratory and Clinical Medicine,1988;111: 267.
[104]Ren MS, Fan YX, Yang RM, et al. A comparative study of biliary elements and clinical phenotypes in Wilson disease. China National Journal of New Gastroenterology,1997;3(4):260-262.
[105]胡文彬,杨任民.肝豆片Ⅰ号对肝豆状核变性患者胆汁微量元素影响的前瞻性研究.中国中西医结合杂志,2001；21(7)：507-509.
[1]Walshe JM. History of Wilson's Disease:1912 to 2000. Movement Disorders, 2006,21(2):142-147.
[2]Ala A, Walker AP, Ashkan K, et al. Wilson's disease. Lancet,2007,369: 397-408.
[3]胡纪源,吕达平,王共强,等.肝豆状核变性的临床误诊研究.中华医学杂志,2001,81(11)：642-644.
[4]Roberts EA, Schilsky ML. A Practice Guideline on Wilson Disease. Hepatology, 2003,37(6):1475-1492.
[5]Bull PC, Thomas GR, Rommens JM, et al. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nature Genetics,1993,5:327-337.
[6]Petrukhin K, Fischer SG, Pirastu M, et al. Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nature Genetics,1993,5:338-343.
[7]Tanzi RE, Petrukhin K, Chernov I, et al. The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nature Genetics,1993,5:344-350.
[8]Ferenci P. Regional distribution of mutations of the ATP7B gene in patients with Wilson disease:impact on genetic testing. Human Genetics,2006,120: 151-159.
[9]Figus A, Lampis R, Devoto M, et al. Carrier detection and early diagnosis of Wilson's disease by restriction fragment length polymorphism analysis. Journal of Medical Genetics,1989,26(2):78-82.
[10]吴志英,王柠,慕容慎行,等.WD基因内部及其两侧翼的微卫星多态快速检出Wilson's病基因携带者及症状前患者.中国神经精神疾病杂志,1996,22(1)：1-3,18,62.
[11]王丽娟,梁秀龄,刘焯霖,等.中国南方汉人D13S316位点多态性及Wilson病基因诊断的研究.中国神经精神疾病杂志,2000,26(2)：90-92.
[12]Gupta A, Maulik M, Nasipuri P, et al. Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers. Clinical Chemistry,2007,53(9):1601-1608.
[13]Harmut H, Schmidt J. Introducing single-nucleotide polymorphism markers in the diagnosis of Wilson disease. Clinical Chemistry,2007,53(9):1568-1569.
[14]Fujita K, Silver J. Single-strand conformational polymorphism. Genome Research,1994,4:137-140.
[15]Thomas GR, Forbes JR, Roberts EA, et al. The Wilson disease gene:spectrum of mutation and their consequences. Nature Genetics,1995;9:210-217.
[16]杨任民,范玉新,余龙,等.肝豆状核变性基因的一种新型错义突变.中华医学杂志,1997；77(5)：344-347.
[17]吴志英,慕容慎行,王柠,等。中国人肝豆状核变性基因第14、18和5号外显子突变及多态研究.临床神经病学杂志,1997,10(5)：259-261.
[18]马少春,徐评议,梁秀龄,等.肝豆状核变性8号14号外显子基因突变的检测.中山医科大学学报,1998,19(1)：14-17,26.
[19]程楠,洪铭范,胡纪源,等.应用双重PCR-SSCP检测Wilson病患者的基因突变.医学研究杂志,2008,37(10)：40-42.
[20]Maier DT, Ferenci P, Polli C, et al. Detection of the His1069Glu mutation in Wilson disease by rapid polymerase chain reaction. Annals of Internal Medicine, 1997,127(1):21-26.
[21]马少春,徐评议,梁秀龄,等.肝豆状核变性8号外显子778密码子基因突变的酶切检测.中国神经精神疾病杂志,1998,24(6)：333-335,381.
[22]王柠,吴志英,林珉婷,等.应用限制性酶谱分析法快速检出Wilson病基因突变热点.中华神经科杂志,1999,32(5)：281-283.
[23]程楠,杨任民,洪铭范,等.PCR-酶切法检测Wilson病935密码子基因突变.中国优生与遗传杂志,2000,8(3)：24-25.
[24]黄帆,梁秀龄,徐评议,等.用荧光PCR对中国人肝豆状核变性进行早期诊断及携带者检测.中华医学遗传学杂志,2001,18(1)：17-20.
[25]Sivakumaran TA, Kucheria K, Oefner PJ. Denaturing high performance liquid chromatography in the molecular diagnosis of genetic disorders. Current Science,2003,84(3):291-296.
[26]Vrabelova S, Letocha O, Borsky M, et al. Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease. Molecular Genetics and Metabolism,2005,86:277-285.
[27]杨静芳,梁秀龄,马守忠,等。74例肝豆状核变性患者中ATP7B基因七种新突变的发现.中华神经科杂志,2006,39(10)：673-677.
[28]Waldenstrom E, Lagerkvist A, Dahlman T, et al. Efficient detection of mutations in Wilson disease by manifold sequencing. Genomics,1996,37: 303-309.
[29]吴志英,王柠,林珉婷,等.Wilson病基因全长外显子的突变检测和分析.中华神经科杂志,2001,34(3)：152-155.
[30]Gresham D, Dunham MJ, Botstein D. Comparing whole genomes using DNA microarrays. Nature Reviews Genetics,2008,9:291-302.
[31]Baner J, Isaksson A, Waldenstrom E, et al. Parallel gene analysis with allele-specific padlock probes and tag microarrays. Nucleic Acids Research, 2003,31(17):103-109.