肝豆状核变性基因表达产物及基因突变的研究

目的 探讨肝豆状核变性（Wilson's disease,WD)的发病机理。方法 将活检获得的WD患者肝标本体外分离,培养肝细胞,应用Western印迹法对WD患者肝细胞WD蛋白进行检测,同时扩增其基因组DNA并直接测序。结果 3例WD患者中有2例出现肝细胞WD蛋白特异条带密度降低。DNA测序发现其中一例患者存在ATP7B778位点CGG→CTG（Arg778Leu)杂合突变及770位点CTC→CTG改变。结论 WD基因在WD患者肝细胞的蛋白表达存在异常。可能与ATP7B基因突变有关。     

Wilson病双(多)胞胎家系的临床和基因突变研究

目的 观察双(多)胞胎Wilson病家系的临床和基因突变特点.方法 收集双(多)胞胎Wilson病家系的临床资料,留取其全血标本,提取基因组DNA,应用短串联重复(short tandem repeats,STR)分型判定双胞胎是否为同卵双生,用DNA测序法检测ATP7B基因各外显子的突变.结果 5个双胞胎家系的患者均符合Wilson病的诊断标准.STR分型提示4个家系为同卵双生,1个家系为异卵双生.3个双胞胎家系的患者均以肝症状起病,另外2个家系的患者以脑症状起病.在4个家系的患者中检出ATP7B基因的突变,均位于第8和(或)第13外显子,其中1个家系的患者同时携带第8外显子p.R778W杂合突变和第13外显子p.P992L纯合突变,其父母分别为p.R778W杂合突变和p.P992L杂合突变的携带者,因此该家系的患者发生了杂合丢失现象.有1个家系的2例患者及其父母亲各外显子均未检出突变.1个三胞胎家系中的1名女性成员为脑症状起病的Wilson病患者,1名男性为无症状的亚临床型Wilson病患者,另1例女性成员未患病,这3位成员及其母亲均检出第13外显子p.P992L杂合突变.结论 本研究结果进一步证实了遗传因素在Wilson病发病中的主要作用.杂合丢失现象是除点突变外Wilson病的另一种发病机制.     

Novel mutations of the ATP7B gene in Japanese patients with Wilson disease

Wilson disease (WD) is an autosomal recessive disorder characterized by copper accumulation in the liver, brain, kidneys, and corneas, and culminating in copper toxication in these organs. In this study, we analyzed mutations of the responsible gene, ATP7B, in four Japanese patients with WD. By direct sequencing, we identified five mutations, of which two were novel, and 16 polymorphisms, of which 6 were novel. The mutations 2871delC and 2513delA shift the reading frame so that truncated abnormal protein is expected. In contrast to these mutations found in patients with hepatic-type of early onset, the mutations A874V, R778L, and 3892delGTC were either missense mutations or inframe 1-amino acid deletion, and occurred in the patients with hepato-neurologic type of late onset. The mutations 2871delC and R778L have been previously reported in a relatively large number of Japanese patients. In particular, R778L is known to be more prevalent in Asian countries than in other countries of the world. Our data are compatible with the hypothesis that the mutations tend to occur in a population-specific manner. Therefore, the accumulation of the types of mutations in Japanese patients with WD will facilitate the fast and effective genetic diagnosis of WD in Japanese patients. Received: October 12, 1999 / Accepted: November 29, 1999     

Clinical presentation and mutations in Danish patients with Wilson disease

This study describes the clinical presentation and diagnosis in all Danish patients (49, 41 unrelated) with Wilson disease (WND). On the basis of the number of diagnosed patients from 1990–2008, the prevalence was estimated to be 1:49 500. Among routinely used diagnostic tests, none were consistently indicative of WND, with the exception of the 24-h urine-Cu test, which is always outside the normal range. Mutations were identified in 100% of the screened ATP7B alleles (70 unrelated), including five novel mutations: p.1021K; p.G1158V; p.L1304F; IVS20-2A>G; Ex5_6del. In all, 70% of mutations were found in exons 8, 14, 17, 18, and 20. The most frequent mutation, p.H1069Q, comprised 18%. We propose a new and simple model that correlates genotype and age of onset. By assuming that the milder of two mutations is ‘functionally dominant'' and determines the age of onset, we classified 25/27 mutations as either severe (age of onset <20 years) or moderate (age of onset >20 years), and correctly predicted the age of onset in 37/39 patients. This method should be tested in other Wilson populations.     

Mutation spectrum and polymorphisms in ATP7B identified on direct sequencing of all exons in Chinese Han and Hui ethnic patients with Wilson's disease

Wilson's disease (WD), an autosomal recessive copper transport disorder, usually presents with symptoms involving the liver or central nervous system. The disease is caused by a large number of mutations in the ATP7B gene comprising 21 expressed exons. Some of the mutations appear to be population specific, whereas others are found in probands from a variety of different ethnic backgrounds. This paper presents the results of screening of the ATP7B gene by means of the direct sequencing of all exons in the gene in 39 Han and one Hui ethnic Chinese patients. Nineteen novel mutations were revealed along with nine others that have been previously described; 57.5% of the mutations were located in exons 8, 13, and 12. In particular, the Arg778Leu mutation in exon 8 was found in 55% of these Chinese patients in at least one allele. Five patients were homozygotes and 17 patients were heterozygotes for Arg778Leu. The detection rate on direct sequencing of the polymerase chain reaction products of all exons of the ATP7B gene in 40 unrelated patients was 83.8% of alleles. Seventeen polymorphisms were also identified in patients and healthy controls. We first reported the presence of ATP7B mutations in Chinese Hui ethnic patients and summarize our results here along with the previously reported findings. A significant correlation between genotype and phenotype was not found in 37 homozygotes and 52 heterozygotes for Arg778Leu.     

Genotyping microarray as a novel approach for the detection of ATP7B gene mutations in patients with Wilson disease

Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism that is caused by mutations in the ATP7B gene. To date, more than 300 mutations have been described in this gene. Molecular diagnostics of WD utilizes restriction enzyme digestion, multiplex ligation-dependent probe amplification or a direct sequencing of the whole gene. To simplify and speed up the screening of ATP7B mutations, we have developed a genotyping microarray for the simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene based on the arrayed primer extension reaction. The patient's DNA is amplified in four multiplex polymerase chain reactions, fragmented products are annealed to arrayed primers spotted on a chip, which enables DNA polymerase extension reactions with fluorescently labeled dideoxynucleotides. The Wilson microarray was validated by screening 97 previously genetically confirmed WD patients. In total, we detected 43 mutations and 15 polymorphisms that represent a majority of the common mutations occurring in the Czech and Slovak populations. All screened sequence variants were detected with 100% accuracy. The Wilson chip appears to be a rapid, sensitive and cost-effective tool, representing the prototype of a disease chip that facilitates and speeds up the screening of potential WD patients.     

中国人WD基因第14和18号外显子的错义突变

目的了解中国人肝豆状核变性（Ｗｉｌｓｏｎ＇ｓｄｉｓｅａｓｅ,ＷＤ）基因第１４和１８号外显子的突变情况,与国外报道的这两个外显子的高突变频率进行比较。方法应用聚合酶链反应－单链构像多态（ＰＣＲ－ＳＳＣＰ）结合ＤＮＡ测序技术,对４４例无亲缘关系的ＷＤ患者及６０例正常对照进行突变检测。结果一例患者在１４号外显子发生了Ａｒｇ１０４１Ｐｒｏ（３１２１Ｇ→Ｃ）纯合错义突变,另一例患者在１８号外显子发生了Ａｓｎ１２７０Ｓｅｒ（３８０９Ａ→Ｇ）杂合错义突变。结论Ａｒｇ１０４１Ｐｒｏ为未报道过的新型错义突变,Ａｓｎ１２７０Ｓｅｒ突变与国外报道一致。但均未呈热区分布。     

Identification of three novel mutations and a high frequency of the Arg778Leu mutation in Korean patients with Wilson disease

Four mutations—R778L, A874V, L1083F, and 2304delC—in the copper-transporting enzyme, P-type ATPase (ATP7B), were identified in Korean Patients with Wilson disease. Arg778Leu, the most frequently reported mutation of this enzyme, was found in six of eight unrelated patients studied, an allele frequency of 37.5%, which is considerably higher than those in other Asian populations. The novel single nucleotide deletion, 2304delC, was found in one patient. Since a mutation at cDNA nucleotide 2302 (2302insC) had been previously described, this region of the ATP7B gene may be susceptible to gene rearrangements causing Wilson disease. Hum Mutat 11:275–278, 1998. © 1998 Wiley-Liss, Inc.     

Spectrum of ATP7B mutations and genotype–phenotype correlation in large‐scale Chinese patients with Wilson Disease

Wilson disease (WD), an inherited disorder associated with ATP7B gene, has a wide spectrum of genotypes and phenotypes. In this study, we developed a rapid multiplex PCR‐MassArray method for detecting 110 mutant alleles of interest, and used it to examine genomic DNA from 1222 patients and 110 healthy controls. In patients not found to have any mutation in the 110 selected alleles, PCR‐Sanger sequencing was used to examine the ATP7B gene. We identified 88 mutations, including 9 novel mutations. Our analyses revealed p.Arg778Leu, p.Arg919Gly and p.Thr935Met showed some correlations to phenotype. The p.Arg778Leu was related to younger onset age and lower levels of ceruloplasmin (Cp) and serum copper, while p.Arg919Gly and p.Thr935Met both indicated higher Cp levels. Besides, the p.Arg919Gly was related to neurological subtype, and p.Thr935Met showed significant difference in the percentage of combined neurological and visceral subtype. Moreover, for ATP7B mutations, the more severe impact on ATP7B protein was, the younger onset age and lower Cp level presented. The feasibility of presymptomatic DNA diagnosis and predicting clinical manifestation or severity of WD would be facilitated with identified mutations and genotype–phenotype correlation precisely revealed in the study.     

Novel ATP7B mutations causing Wilson disease in several Israeli ethnic groups

We characterized microsatellite marker haplotypes and identified mutations in members of 19 ethnically diverse Israeli families affected by Wilson disease (WD). Eighteen unique haplotypes were derived from allelic combinations for four marker loci spanning the WD gene, ATP7B, at chromosome 13q14.3: D13S133, D13S296, D13S301 and D13S295. Most of these haplotypes are population specific and vary among and even within different ethnic groups. Intrafamilial variability of WD haplotypes was observed in two large consanguineous families in which a single origin of WD was expected. In contrast, some WD haplotypes were identified in more than one group. Five novel and four previously described mutations were detected in our sample. The novel mutations include two deletions (845delT and 1639delC) and three missense mutations (E1064A, M645R, and G1213V). Mutations were identified for 11 of the 18 WD haplotypes, suggesting that other mutations may reside in noncoding regions of the ATP7B gene. Identification of all WD mutations will undoubtedly increase our understanding of the normal function of ATP7B as well as lead to more accurate prognosis and genetic counseling. Hum Mutat 11:145–151, 1998. © 1998 Wiley-Liss, Inc.     

Identification of novel ATP7B gene mutations and their functional roles in Korean patients with Wilson disease

Wilson disease (WND), an autosomal recessive disorder of copper transport, is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues because of impaired biliary copper excretion and disturbed incorporation of copper into ceruloplasmin. Hepatic cirrhosis and neuronal degeneration are the major symptoms of WND, and mutations in the ATP7B gene are associated with WND. We have identified 28 different mutations in the ATP7B gene, including six novel variations, in 120 unrelated Korean patients with WND. Molecular defects in ATP7B were present in only 75.0% of Korean WND patients, with the most common mutation, p.Arg778Leu, having an allele frequency of 39.2%. To evaluate the functional defects of ATP7B caused by novel mutations, we used a yeast complementation system, and we used confocal microscopy to localize each mutation after transient expression in mammalian cells. Six novel variations were cloned into a yeast expression vector and two into a mammalian expression vector for confocal analysis. We found that c.2785A>G (p.Ile929Val) and c.3316G>A (p.Val1106Ile) were rare polymorphisms, whereas the others were novel variations disturbing ATP7B function.     

Functional analysis of mutations in the ATP loop of the Wilson disease copper transporter,ATP7B

Wilson disease (WND) is an autosomal recessive disorder resulting from mutation of ATP7B. Transport of copper by ATP7B from the trans‐Golgi of hepatocytes into apical membrane‐trafficked vesicles for excretion in the bile is the major means of copper elimination from the body. Although copper is an essential nutrient, homeostasis must be carefully maintained. If homeostasis is disrupted, copper can accumulate within the liver, kidney, cornea, and/or brain. The range of organs affected leads to clinical heterogeneity and difficulty in WND diagnosis. Sequencing of ATP7B is an important adjunct for diagnosis but has led to the discovery of many novel missense variants. Although prediction programs are available, functional characterization is essential for determining the consequence of novel variants. We have tested 12 missense variants localized to the ATP loop of ATP7B and compared three predictive programs (SIFT, PolyPhen, and Align‐GVGD). We found p.L1043P, p.G1000R, p.G1101R, p.I1102T, p.V1239G, and p.D1267V deleterious; p.G1176E and p.G1287S intermediate; p.E1173G temperature sensitive; p.T991M and p.I1148T mild; and p.R1228T functioning as wild type. We found that SIFT most often agreed with functional data (92%), compared with PolyPhen (83%) and Align‐GVGD (67%). We conclude that variants found to negatively affect function likely contribute to the WND phenotype in patients. Hum Mutat 31:569–577, 2010. © 2010 Wiley‐Liss, Inc.     

Common mutations of ATP7B in Wilson disease patients from Hungary

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. The H1069Q mutation in exon 14 of ATP7B is far the most frequent in Wilson patients of European origin. Mutations in exon 8 and 15 are also common among the over 150 described mutations in the WD gene. The aim was to investigate the frequency of these common WD gene mutations in Hungarian patients. A total of 42 patients with WD from 39 Hungarian families were examined. The H1069Q mutation was assessed by a seminested polymerase chain reaction (PCR)‐based restriction fragment length polymorphism (RFLP) assay, while mutations in exons 8, 13, 15, and 18 of WD gene were identified by sequencing. In addition, haplotype analysis was performed using three common microsatellite markers (D13S314, D13S301, D13S316). The H1069Q mutation was found in 27 patients (64.3%). Nine patients were H1069Q homozygous. Eighteen patients were H1069Q compound heterozygous, two of them had H1069Q/P969Q and one patient H1069Q/3400delC genotype. In two of the 15 H1069Q‐negative patients a novel mutation in exon 13 (T977M) was detected. One H1069Q‐negative patient had a mutation in exon 8 (G710S). None of the studied mutations was detected in 12 WD patients. H1069Q‐positive patients from various European countries had the same haplotype pattern. The H1069Q point mutation is frequent in Hungarian patients with WD and appears to have originated from a single founder in Eastern Europe. In contrast, mutations in exons 8, 13, 15, and 18 are uncommon in Hungarian WD patients. © 2002 Wiley‐Liss, Inc.     

Sequence variation database for the Wilson disease copper transporter, ATP7B

Wilson disease (WND) is a disorder of copper transport resulting in copper accumulation in liver, kidney, and brain. This recessive disorder expresses variable clinical symptoms affecting liver, brain, and/or kidney. The age of onset of symptoms varies from 3 to almost 70 years, so the diagnosis for this treatable disorder is easily missed. The defective gene is a membrane P-type ATPase, with similar structure to the other metal transporting ATPases. Most patients with Wilson disease are compound heterozygotes. This report describes the database we have developed for reporting of mutations in ATP7B, the gene defective in WND. The database includes more than 518 variants (379 probable disease-causing and the remainder possible normal variants) from populations worldwide (Available at: www.medicalgenetics.med.ualberta.ca/wilson/index.php; Last accessed: 20 June 2007). The tables in this database are a valuable resource for the study of population variation and the function of the transporter, and will assist in the identification of disease and non-disease-causing sequence variants.     

应用高分辨熔解曲线技术检测中国人Wilson病的常见基因突变

目的 应用PCR-高分辨熔解曲线分析技术检测中国人Wilson病(Wilson disease,WD)患者中ATP7B基因内高频突变区第8和13外显子.方法 酚-氯仿法提取外周全血基因组DNA;PCR扩增患者ATP7B基因第8和13外显子及两个外显子中的突变热点区域,PCR产物经HR-1进行高分辨熔解曲线分析;进一步以限制性内切酶或(和)DNA测序法对高分辨熔解曲线检测结果进行验证.结果 在30例WD患者中,检测到R778L纯合子3例,R778L杂合子6例,P992L杂合子6例,P992D/S975Y的复合杂合子1例,R778L/P992L复合杂合子2例和R778L/752.33delG复合杂合子1例.本组样本中,R778L、P992L和S975Y的基因频率分别为25%、15%和1.67%.测序及限制性内切酶分析结果完全与高分辨熔解曲线分析结果一致.结论 高分辨熔解曲线分析检测ATP7B基因突变具有简便、快速、特异和灵敏等优点,可作为Wilson病患者及携带者突变筛查的优选方法.     

Functional characterization of novel or yet uncharacterized ATP7B missense variants detected in patients with clinical Wilson's disease

Wilson's disease (WD, MIM#277900) is an autosomal recessive disorder resulting in copper excess caused by biallelic variants in the ATP7B gene (MIM#606882) encoding a copper transporting P-type ATPase. ATP7B variants of unknown significance (VUS) are detected frequently, sometimes impeding a clear diagnosis. Functional analyses can help to classify these variants as benign or pathogenic. Additionally, variants already classified as (likely) pathogenic benefit from functional analyses to understand their pathomechanism, thus contribute to the development of personalized treatment approaches in the future. We described clinical features of six WD patients and functionally characterized five ATP7B missense variants (two VUS, three yet uncharacterized likely pathogenic variants), detected in these patients. We determined the protein level, copper export capacity, and cellular localization in an in vitro model and potential structural consequences using an ATP7B protein model based on AlphaFold. Our analyses give insight into the pathomechanism and allowed reclassification for the two VUS to likely pathogenic and for two of the three likely pathogenic variants to pathogenic.     

Value of histochemical stains for copper in the diagnosis of Wilson's disease

Aims : The histochemical demonstration of hepatic copper is important in the diagnosis of Wilson's disease (WD). Conflicting results have been published with regard to the ability of different histochemical methods to demonstrate copper storage in the liver. Therefore, we evaluated the diagnostic value of three available histochemical methods in a large series of patients affected by WD.  

Methods and results

Seventy-nine consecutive liver needle biopsies, from 74 patients, 39 males and 35 females, aged 4–60 years (mean age 28.5 years) were stained with orcein, rhodanine and using Timm's method. On the basis of the histological picture, liver biopsies were subdivided into three groups: group A, steatosis; group B, interface hepatitis; group C, chronic hepatitis with bridging fibrosis and/or cirrhosis. In group A, 30.4% of the cases were positive using Timm's method, vs 13.2% using the rhodanine and 17.5% using the orcein method. In group B, Timm's method was positive in 40.1% while rhodanine and orcein showed positivity in 26.7%. In group C, the Timm's method stained 58.6%, rhodanine 36.6% and orcein 29.3% positively.  

Conclusions

Our data show that: (1) Timm's silver stain is the most sensitive method for the demonstration of copper in all cases of WD; (2) rhodanine and orcein have minor value in the diagnosis of WD, especially in the early stages of the disease; (3) to increase the diagnostic value of histochemistry for copper multiple histochemical stains in serial sections are required; and (4) although hepatic copper concentration is highest in the early stages of WD, the histochemical demonstration fails in a large number of cases.     

Identification and characterization of 15 novel GALC gene mutations causing Krabbe disease

The characterization of the underlying GALC gene lesions was performed in 30 unrelated patients affected by Krabbe disease, an autosomal recessive leukodystrophy caused by the deficiency of lysosomal enzyme galactocerebrosidase. The GALC mutational spectrum comprised 33 distinct mutant (including 15 previously unreported) alleles. With the exception of 4 novel missense mutations that replaced evolutionarily highly conserved residues (p.P318R, p.G323R, p.I384T, p.Y490N), most of the newly described lesions altered mRNA processing. These included 7 frameshift mutations (c.61delG, c.408delA, c.521delA, c.1171_1175delCATTCinsA, c.1405_1407delCTCinsT, c.302_308dupAAATAGG, c.1819_1826dupGTTACAGG), 3 nonsense mutations (p.R69X, p.K88X, p.R127X) one of which (p.K88X) mediated the skipping of exon 2, and a splicing mutation (c.1489+1G>A) which induced the partial skipping of exon 13. In addition, 6 previously unreported GALC polymorphisms were identified. The functional significance of the novel GALC missense mutations and polymorphisms was investigated using the MutPred analysis tool. This study, reporting one of the largest genotype-phenotype analyses of the GALC gene so far performed in a European Krabbe disease cohort, revealed that the Italian GALC mutational profile differs significantly from other populations of European origin. This is due in part to a GALC missense substitution (p.G553R) that occurs at high frequency on a common founder haplotype background in patients originating from the Naples region.     

高分辨熔解曲线技术在Wilson病致病基因突变鉴定中的应用

目的在一个肝豆状核变性(WD)家系中进行致病突变鉴定和产前基因诊断。方法酚-氯仿法提取外周全血或妊娠13周胎儿绒毛组织基因组DNA;利用PCR-Sanger测序技术对先证者ATP7B基因外显子及外显子/内含子衔接区序列进行突变分析;针对先证者携带的突变位点,应用聚合酶链反应(PCR)-高分辨熔解曲线(HRM)方法对先证者家系4名成员进行突变鉴定,并在此基础上为患儿母亲提供产前基因诊断。结果先证者ATP7B基因第8外显子存在纯合致病突变c.2333GT(p.R778L);该家系5名成员和4名群体正常样本分属于3种不同熔解曲线。HRM分析结果与测序结果一致,即:患儿本人为R778L的纯合子,其父母、姐姐和母亲产前诊断的胎儿均为突变杂合子,4名群体正常对照为纯合野生型。结论在一个WD家系中检测到ATP7B基因热点突变c.2333GT(p.R778L),并针对该突变建立了一种基于PCR-HRM技术的快速突变鉴定方法,成功为家系提供产前基因诊断。     

Genetic variation in the promoter and 5' UTR of the copper transporter, ATP7B, in patients with Wilson disease

ATP7B is a copper-transporting P-type ATPase defective in the copper transport disorder, Wilson disease (WND). We have sequenced the 5' UTR and promoter region of ATP7B in 37 unrelated WND patients in whom partial sequencing of the coding region and intron/exon boundaries of the gene had failed to identify one or both disease-causing mutations. Three patients were found to be heterozygous for a 15 bp deletion between nucleotides -424 and -441. This deletion had been previously identified as the most common mutation in Sardinian WND patients. Two novel single-nucleotide changes were also identified within the 5' UTR and promoter of ATP7B; however, these were found at a similar frequency in control chromosomes and are apparently normal variants. These results suggest that mutations in regulatory elements of ATP7B are uncommon in patients of European ancestry, except in Sardinia.