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.     

Six novel ATP7B mutations in Thai patients with Wilson disease

WD is an autosomal recessive disorder of copper transport resulting in excessive copper deposition in the liver and brain. It is caused by defects of ATP7B encoding a copper transporting P-type ATPase. To identify the mutations in ATP7B in Thai patients with WD, DHPLC analysis was applied to detect mutations and polymorphisms of the entire ATP7B gene in 19 Thai patients with WD. Mutations in ATP7B were identified in 14 of 19 patients: 2 homozygotes, 8 compound heterozygotes and 4 heterozygotes. Eighteen mutations distributed throughout the entire coding region of ATP7B gene including 11 missense, 3 nonsense, 1 splice-site, 1 deletion and 2 insertions. Of 18 different mutations identified, 6 were found to be novel. Twelve single nucleotide polymorphisms (SNPs) were also identified and two SNPs have not yet previously been reported. Segregation analysis using DHPLC analysis showed mutation transmission patterns within each family of Thai patients with WD. Mutations in ATP7B in Thai patients with WD are worth adding into the public database for genetic epidemiology and population genetics.     

ATP7B variant spectrum in a French pediatric Wilson disease cohort

Background/aimThe spectrum of ATP7B variants varies significantly according to geographic distribution, and there is insufficient data on the variants observed in the French population.MethodsClinical data of 113 children included in the French WD national registry were gathered from March 01, 1995 to July 01, 2020. Data included epidemiological, clinical, laboratory, genetics.ResultsDiagnosis was made at a mean age of 11.0 ± 4.1 years (range 1–18 years). At diagnosis, 91 patients (79.8 %) had hepatic manifestations, 18 (15.8 %) presented neurological manifestations, and 4 patients (3.5 %) were asymptomatic. Only 29 patients (25 %) were homozygous for a variant. We have found a total of 102 different variants including 14 novel variants. Recurrent variant p.His1069Gln was the most prevalent, n = 31 alleles (14,2%), with only seven homozygous; in contrast 55% of variants are identified in only one family. 45% were truncating variants. In respect of mutated exon, the three most prevalent were exon 14 (16.5%), exon 8 (13.8%), and exon 3 (11.5%). When considering patients with two Nonsense / Frameshift variants as a group and those with two Missense variants, we found significantly lower ceruloplasmin for the former: 2.8 ± 0.7 mg/dl vs 8.4 ± 5mg/dl (p<0.05).Conclusionp.His1069Gln is the most frequent variant (14,2%) and exons 14, 8, and 2 of the ATP7B gene account for 41.7% of total variants. However, there is significant heterogeneity in the French population concerning the other ATP7B variants. Nonsense / Frameshift variants were associated with lower ceruloplasmin levels.     

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.     

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.     

New mutations and polymorphisms of the ATP7B gene in sporadic Wilson disease

Wilson's disease (WD) is a rare autosomal recessive genetic disorder of copper metabolism resulting in brain damage, liver failure, and neurological impairment and psychiatric disturbances, as a result of excessive copper accumulation in the brain, liver, kidneys and eyes. ATP7B, encoding a copper transporter P-ATPase was identified as the causative gene of WD. Mutations in the ATP7B gene lead to the defection of the transmembrane transporter so that it can not metabolize copper effectively. We reported the clinical and molecular features of three unrelated and non-consanguineous WD patients. We performed molecular genetic analysis of the ATP7B gene in all cases by DNA sequencing, and revealed 7 novel single nucleotide polymorphisms (SNPs) and 8 well known mutations. Among them, that novel SNP (c. -520 C>T) and two well known mutations (c. 2310 C>G/p. Leu700Leu, c. 2333 G>T/A/p. Arg778Leu/Gln) coexisted in all patients and they were heterozygous and homozygous in the youngest case, respectively, indicating that they may be correlated to the pathogenesis and potentially used as a genetic biomarker for early WD diagnosis.     

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     

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.     

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.     

Copper-induced translocation of the Wilson disease protein ATP7B independent of Murr1/COMMD1 and Rab7

Wilson disease is a genetic disorder of copper metabolism. Impaired biliary excretion results in a gradual accumulation of copper, which leads to severe disease. The specific gene defect lies in the Wilson disease protein, ATP7B, a copper-transporting ATPase that is highly active in hepatocytes. The two major functions of ATP7B in the liver are the copper loading of ceruloplasmin in the Golgi apparatus, and the excretion of excess copper into the bile. In response to elevated copper levels, ATP7B shows a unique intracellular trafficking pattern that is required for copper excretion from the Golgi apparatus into dispersed vesicles. We analyzed the translocation of ATP7B by both confocal microscopy and RNA interference, testing current models that suggest the involvement of Murr1/COMMD1 and Rab7 in this pathway. We found that although the ATP7B translocation is conserved among nonhepatic cell lines, there is no co-localization with Murr1/COMMD1 or the Rab marker proteins of the endolysosomal system. Consistent with this finding, the translocation of ATP7B was not impaired by the depletion of either Murr1/COMMD1 or Rab7, or by a dominant-negative Rab7 mutant. In conclusion, our data suggest that the translocation of ATP7B takes place independently of Rab7-regulated endosomal traffic events. Murr1/COMMD1 plays a role in a later step of the copper excretion pathway but is not involved in the translocation of the Wilson disease protein.     

Mutational analysis of ATP7B gene in Egyptian children with Wilson disease: 12 novel mutations

The aim of this work was to study the mutations within ATP7B in Egyptian children with Wilson disease and to evaluate any potential correlation between genotype and phenotype in this cohort. The study consisted of 48 children with Wilson disease from 32 independent families. The 21 exons of the ATP7B gene were amplified in a thermal cycler. Direct sequencing of the amplified polymerase chain reaction (PCR) products was performed by cycle sequencing using fluorescent dye terminators in an automatic ABI sequencer. Thirty-one different mutations in 96 chromosomes were detected (19 missense, three nonsense, seven frameshift deletions, and two splice-site mutations). Of these, 12 mutations have not been previously reported. The p.N1270S, p.C703Y, IVS18-2A > G, p.R1319X, c.2304-2305insC, and p.H1069Q were present in 7.8%, 6.2%, 6.2%, 6.2%, 4.7%, and 4.7%, respectively, of studied chromosomes in independent families. One patient was homozygous for both p.N1270S and p.T1434M mutations. Frameshift and nonsense mutations were found in 50% of patients with disease onset < or =8 years compared with only 26% in patients with onset >8 years. Despite mutation heterogeneity in Egyptian children, genotype-phenotype correlation analysis seems to be promising in this population, as many patients carry homozygous mutations, a situation that mandates a larger-scale population screening to identify the carrier rate in this community.     

Wilson disease: novel mutations in the ATP7B gene and clinical correlation in Brazilian patients

Wilson disease (WD) is a rare inherited autosomal recessive disorder caused by a defect in a metal transporting P-type ATPase, resulting in copper overload in various tissues and cells. The aim was to assess both the phenotype in Brazilian WD patients and the corresponding ATP7B genotype. Sixty subjects belonging to 46 pedigrees diagnosed as WD were included in this study. Direct sequencing of all 21 exons within ATP7B and their flanking introns was performed. Demographic, clinical, laboratory and histopathological data at the time of diagnosis were obtained. We identified twenty-five mutations, twelve of them reported for the first time. The c.3402delC mutation had the highest allelic frequency (30.8%), followed by the c.2123T>C (p.L708P) (16.7%). Exons 8 and 15 were the site of 62.5% of the mutations. The common European mutation c.3207C>A (p.H1069Q) was not present at all. Phenotype varied greatly among individuals with the same ATP7B genotype. Our data confirm the heterogeneity of ATP7B genotype in Brazilian WD patients. The mutational spectrum is compatible with the Brazilian history of Mediterranean immigration; however, new mutations, and different frequencies and phenotype associated with the previously known mutations characterize this population. Exons 8 and 15 should be preferentially screened in WD cases from Brazil. Phenotype variation among subjects with the same ATP7B genotype suggests that modifying factors play an additional role in the pathogenesis of WD.     

Mutation analysis of ATP7B gene in Turkish Wilson disease patients: Identification of five novel mutations

Wilson disease is an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene that encodes a P-type copper transporting ATPase. The aim of this study was to screen and detect mutations of the ATP7B gene in unrelated Turkish Wilson disease patients (n = 46) and control group (n = 52). Mutations were screened and detected by DNA sequencing. 30 out of 46 patients had mutations. 24 different Wilson disease related mutations were identified in those patients. The distribution of mutations in ATP7B gene was as follow: 17 missense, 3 nonsense, 1 silent, 3 frameshift (1 insertion, 2 deletion). None of them were not found in the control group. Five out of 24 mutations were found to be novel. Four of them were missense (c.2363C > T, c.3106G > A, c.3451C > T, c.3733C > A). The last one was deletion (c.3111delC). 10 single nucleotide polymorphisms (SNPs) given in the literature were found in both control and patients groups. Moreover one new polymorphism in exon 18 (c.3727G > A) not reported previously was discovered in both groups. It was striking that most of the mutations were found in exons 8, 12–14. This is the first study covering Turkish Wilson disease patients and control groups for mutation screening in all the coding regions of ATP7B gene by DNA sequencing method and adding five new mutations and one polymorphism into the HUGO Wilson disease mutation database.     

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.     

Genotype-phenotype correlations for a wide spectrum of mutations in the Wilson disease gene (ATP7B)

Wilson disease (WND) is caused by mutations in the ATP7B gene and exhibits substantial allelic heterogeneity. In this study we report the results of molecular analyses of 20 WND families not described previously. When combined with our prior results, the cohort includes 93 index patients from 69 unrelated families. Twenty different mutations accounted for 86% of the WND chromosomes. The most frequent were p.H1069Q (35%), p.R969Q (12%), c.2530delA (7%), p.L936X (7%), p.Q289X (7%), and p.I1148T (3%). We also present here a detailed phenotypic assessment for patients whose molecular result was previously reported. Thirty cases were homozygous for 9 different mutations, 13 of which were homozygous for p.H1069Q, and 7 for p.R969Q. Mutations p.H1069Q and p.R969Q appeared to confer a milder disease as patients showed disease onset at a later age, and were associated with milder severity when found in trans with severe mutations. Predicted nonsense and frameshift mutations were associated with severe phenotypic expression with earlier disease onset and lower ceruloplasmin values. WND can be treated by copper-chelation therapy, particularly if the disease is diagnosed before irreversible tissue damage occurs. Our results on the effect of predicted nonsense and frameshift mutations are especially important for early medical intervention in presymptomatic infants and children with these genotypes.     

The Wilson disease protein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease.     

中国人Wilson病ATP7B基因Thr935Met突变与临床表型的关系研究

目的　探讨Wilson病 (Wilsondisease,WD)ATP7B基因突变的高频位点之一Thr 935Met与临床表现的关系。方法　应用PCR技术扩增 90例WD患者和 30例正常人ATP7B基因第 12外显子 ,其PCR产物行限制性内切酶TaiI酶切分析。结果　正常人组酶切未见异常。 90例WD组第 12外显子有 10例Thr 935Met杂合突变 ,未见纯合突变 ,检出率为 11 1%。突变组的平均发病年龄为 19 10岁± 8 5 2岁 ,无突变组的平均发病年龄为 14 0 0岁± 7 4 2岁 ,差异有显著意义 (P =0 0 4 7)。结论　Thr 935Met突变组患者的发病年龄迟于未见该点突变组的患者 ,而性别、首发症状及铜生化水平与该点突变无明显相关性。