Clusterin and COMMD1 independently regulate degradation of the mammalian copper ATPases ATP7A and ATP7B

ATP7A and ATP7B are copper-transporting P(1B)-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis.     

Twenty-four novel mutations in Wilson disease patients of predominantly Italian origin

Herein we report the results of mutation analysis of the ATP7B gene in a group of 134 Wilson disease (WD) families (268 chromosomes) prevalently of Italian origin. Using the SSCP and sequencing methods we identified 71 disease-causing mutations. Twenty-four were novel, while 19 more mutations already described, were identified in new populations in this study. A known mutation G591D showed a regional distribution, since it was only detected in 38.5% of the analyzed chromosomes in WD patients originating from Apulia, a region of South Italy. Detection of new mutations in the ATP7B gene increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.     

Mutation analysis of Taiwanese Wilson disease patients

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, which is caused by mutation in copper-transporting ATPase (ATP7B). In the present study, we report a molecular diagnosis method to screen the WD chromosome in patients or in heterozygotic carriers in Taiwan. Exons 8, 11, 12, 13, 16, 17, and 18 of ATP7B are selected for the screening of mutations. The most common mutation, Arg778Leu or Arg778Gln, was first screened by PCR-RFLP then we combined single-stranded conformation polymorphism (SSCP) analysis followed by direct DNA sequencing on the DNA fragments with mobility shift on SSCP analysis. The diagnostic rate was compared with standard ATP7B whole gene sequencing analysis. Ten different mutations were identified among 29 WD patients; among them four were novel (Ala1168Pro, Thr1178Ala, Ala1193Pro, and Pro1273Gln). The false positive rates were tested against 100 normal individuals and listed as follows: exon 8: 5%; exon 11: 4%; exon 12: 6%; exon 13: 5%; exon 16: 5%; exon 17: 3%; exon 18: 4%. The Arg778Leu mutation exhibited the highest allelic frequency (43.1%). The detection rate of WD chromosomes is 65.52%, which is as sensitive as whole gene sequencing scanning. According to our results, WD chromosomes in Taiwan are predominantely located at exons 8, 11, 12, 13, 16, 17, and 18. The standard sequencing analysis on the entire gene is time consuming. We recommend screening these 7 exons first on those individuals who have a higher risk in having WD, before whole gene and promoter sequencing analysis in Taiwan.     

Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotype-phenotype correlation, and functional analyses.

Wilson disease (WD) is an autosomal recessive disorder characterized by toxic accumulation of copper in the liver and subsequently in the brain and other organs. On the basis of sequence homology to known genes, the WD gene (ATP7B) appears to be a copper-transporting P-type ATPase. A search for ATP7B mutations in WD patients from five population samples, including 109 North American patients, revealed 27 distinct mutations, 18 of which are novel. A composite of published findings shows missense mutations in all exons-except in exons 1-5, which encode the six copper-binding motifs, and in exon 21, which spans the carboxy-terminus and the poly(A) tail. Over one-half of all WD mutations occur only rarely in any population sample. A splice-site mutation in exon 12 accounts for 3% of the WD mutations in our sample and produces an in-frame, 39-bp insertion in mRNA of patients homozygous, but not heterozygous, for the mutation. The most common WD mutation (His1069Glu) was represented in approximately 38% of all the WD chromosomes from the North American, Russian, and Swedish samples. In several population cohorts, this mutation deviated from Hardy-Weinberg equilibrium, with an overrepresentation of homozygotes. We did not find a significant correlation between His1069Glu homozygosity and several clinical indices, including age of onset, clinical manifestation, ceruloplasmin activity, hepatic copper levels, and the presence of Kayser-Fleischer rings. Finally, lymphoblast cell lines from individuals homozygous for His1069Glu and 4 other mutations all demonstrated significantly decreased copper-stimulated ATPase activity.     

COMMD1, a multi-potent intracellular protein involved in copper homeostasis,protein trafficking,inflammation, and cancer

Copper is a trace element indispensable for life, but at the same time it is implicated in reactive oxygen species formation. Several inherited copper storage diseases are described of which Wilson disease (copper overload, mutations in ATP7B gene) and Menkes disease (copper deficiency, mutations in ATP7A gene) are the most prominent ones. After the discovery in 2002 of a novel gene product (i.e. COMMD1) involved in hepatic copper handling in Bedlington terriers, studies on the mechanism of action of COMMD1 revealed numerous non-copper related functions. Effects on hepatic copper handling are likely mediated via interactions with ATP7B. In addition, COMMD1 has many more interacting partners which guide their routing to either the plasma membrane or, often in an ubiquitination-dependent fashion, trigger their proteolysis via the S26 proteasome. By stimulating NF-κB ubiquitination, COMMD1 dampens an inflammatory reaction. Finally, targeting COMMD1 function can be a novel approach in the treatment of tumors.     

Characterization of the molecular defect in the ATP7B gene in Wilson disease patients from Yugoslavia

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism resulting from the absence or dysfunction of a copper transporting P-type ATPase (ATP7B). Approximately 150 mutations of the ATP7B have been identified to date. In this paper, we report the results of molecular characterization and genotype-phenotype analysis, which we have carried out on 35 patients from Yugoslavia affected by WD. Using single-strand conformational polymorphism (SSCP) followed by direct sequencing, we characterized the molecular defect in 80% of WD chromosomes and found 11 different mutations, three of which are novel. The most common mutations that accounted for the molecular defect in 71.3% of WD chromosomes were H1069Q (48.9%), 2304-2305insC (11.4%), R616Q (5.7%), and A1003T (5.7%). The results produced in this paper indicate that the best strategy for mutation detection in Yugoslavian patients with WD is an SSCP analysis of exons 14, 8, 5, and 13, where most of the defects (73.1%) lie, followed by mutation analysis of the remaining exons in ATP7B in patients in whom the mutation was not detected by the finitial screening. These data can be used to develop straightforward genetic testing in this population or in other countries composed of a genetically mixed population like the United States, where a significant number of immigrants came from Central and Eastern Europe.     

Molecular Pathogenesis of Wilson Disease Among Indians: A Perspective on Mutation Spectrum in ATP7B gene, Prevalent Defects, Clinical Heterogeneity and Implication Towards Diagnosis

Aims We aim to identify the molecular defects in the ATP7B, the causal gene for Wilson disease (WD), in eastern Indian patients and attempt to assess the overall mutation spectrum in India for detection of mutant allele for diagnostic purposes. Methods Patients from 109 unrelated families and their first-degree relatives comprising 400 individuals were enrolled in this study as part of an ongoing project. Genomic DNA was prepared from the peripheral blood of Indian WD patients. PCR was done to amplify the exons and flanking regions of the WD gene followed by sequencing, to identify the nucleotide variants. Results In addition to previous reports, we recently identified eight mutations including three novel (c.3412 + 1G > A, c.1771 G > A, c.3091 A > G) variants, and identified patients with variable phenotype despite similar mutation background suggesting potential role of modifier locus. Conclusions So far we have identified 17 mutations in eastern India including five common mutations that account for 44% of patients. Comparative study on WD mutations between different regions of India suggests high genetic heterogeneity and the absence of a single or a limited number of common founder mutations. Genotype–phenotype correlation revealed that no particular phenotype could be assigned to a particular mutation and even same set of mutations in different patients showed different phenotypes.     

The spectrum of pathogenic variants of the ATP7B gene in Wilson disease in the Russian Federation

Background. Wilson’s disease (WD) is a rare inherited disorder caused by mutations in the ATP7B gene resulting in copper accumulation in different organs. However, data on ATP7B mutation spectrum in Russia and worldwide are insufficient and contradictory. The objective of the present study was estimation of the frequency of ATP7B gene mutations in the Russian population of WD patients. Materials and methods. 75 WDpatients were examined by next-generation sequencing (NGS). A targeted panel NimbleGen SeqCap EZ Choice: 151012_HG38_CysFib_EZ_HX3 (ROCHE)was designed for analysis of ATP7B gene and possible modifier genes. Retrospective assessment of a diagnostic WD score (Leipzig, 2001) was also performed. Results. 31 mutations in ATP7B gene were detected. Two most frequent mutations were c.3207C > A (51,85% of alleles) and c.3190 G > A (8,64% of alleles). Single rare mutations were detected in 29% of cases. In 96% cases mutations of both copies of the ATP7B were revealed. We also observed 3 novel potentially pathogenic variants which were not previously described (c.1870-8A > G, c.3655A > T (p.Ile1219Phe), c.3036dupC (p.Lys1013fs). For 25% of patients at the time of the manifestation the diagnosis WD could not be established using the earlier proposed diagnostic score. There was a remarkable delay in diagnosis for the majority of patients. Only 33% of patients WD was diagnosed in three months after the first symptoms, 29%patients - in 3–12 months, 30% – in 1–10 years, in 8% – more than 10 years. Generally, clinical appearance of WD may be rather variable at manifestation and genetic profiling at this step is the only way to confirm the presence of WD.     

Determinants for Simultaneous Binding of Copper and Platinum to Human Chaperone Atox1: Hitchhiking not Hijacking

Cisplatin (CisPt) is an anticancer agent that has been used for decades to treat a variety of cancers. CisPt treatment causes many side effects due to interactions with proteins that detoxify the drug before reaching the DNA. One key player in CisPt resistance is the cellular copper-transport system involving the uptake protein Ctr1, the cytoplasmic chaperone Atox1 and the secretory path ATP7A/B proteins. CisPt has been shown to bind to ATP7B, resulting in vesicle sequestering of the drug. In addition, we and others showed that the apo-form of Atox1 could interact with CisPt in vitro and in vivo. Since the function of Atox1 is to transport copper (Cu) ions, it is important to assess how CisPt binding depends on Cu-loading of Atox1. Surprisingly, we recently found that CisPt interacted with Cu-loaded Atox1 in vitro at a position near the Cu site such that unique spectroscopic features appeared. Here, we identify the binding site for CisPt in the Cu-loaded form of Atox1 using strategic variants and a combination of spectroscopic and chromatographic methods. We directly prove that both metals can bind simultaneously and that the unique spectroscopic signals originate from an Atox1 monomer species. Both Cys in the Cu-site (Cys12, Cys15) are needed to form the di-metal complex, but not Cys41. Removing Met10 in the conserved metal-binding motif makes the loop more floppy and, despite metal binding, there are no metal-metal electronic transitions. In silico geometry minimizations provide an energetically favorable model of a tentative ternary Cu-Pt-Atox1 complex. Finally, we demonstrate that Atox1 can deliver CisPt to the fourth metal binding domain 4 of ATP7B (WD4), indicative of a possible drug detoxification mechanism.     

The copper chaperone Atox1 in canine copper toxicosis in Bedlington terriers

Copper toxicosis, resulting in liver disease, commonly occurs in Bedlington terriers. This recessively inherited disorder, similar in many respects to Wilson disease, is of particular interest because the canine Atp7b gene, homologous to ATP7B defective in Wilson disease, is not responsible for canine copper toxicosis as has been expected. Atox1, a copper chaperone delivering copper to Atp7b, therefore became a potential candidate. We cloned canine Atox1, which shows conserved motifs of the copper-binding domain (MTCXXC) and of the lysine-rich region (KTGK), and showed 88, 80, and 41% amino acid sequence identity with the orthologous mouse, human, and yeast proteins. No gross deletions of Atox1 could be identified in the affected Bedlington terriers by Southern blot analysis of genomic DNA. The canine Atox1 gene spans about 4 kb, with a 204-bp open reading frame cDNA contained within two exons. Sequence analysis of the coding regions, including intron/exon boundaries, showed no mutations in Atox1 from genomic DNA of an affected dog. We have also identified an apparently nontranscribed canine Atox1 pseudogene, with 12 sequence changes and no intron. Mapping of Atox1 and a marker closely linked to the canine copper toxicosis locus indicated lack of synteny. Atox1 is therefore excluded as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene must be responsible for this copper storage disease in dogs and also suggesting the possibility of a similar gene responsible for a copper storage disease in humans.     

From clinical and biochemical to molecular genetic diagnosis of Wilson disease in Latvia

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism characterized by hepatic and/or neurological damage. More than 300 mutations in gene ATP7B causing this defect have been reported. The data on correlation between WD patient genotypes and clinical presentation are controversial. In this paper, the results of ATP7B mutation analysis by testing for mutation H1069Q and direct sequencing of six exons together with the clinical data of 40 Latvian WD patients are presented. Two previously described and two novel mutations as well as one previously reported polymorphism were identified. The H1069Q mutation was present at 52.5% of the disease alleles. One individual among 157 healthy Latvians was also found to be a mutation H1069Q carrier. The estimated incidence of WD in Latvia is ∼1 in 25600. Wide clinical variability was observed among individuals with the same ATP7B genotype, thus supporting the suggestion that modifying factors play an additional role in the pathogenesis of WD. An algorithm for the diagnosis of WD, including testing for mutation H1069Q, is recommended for the populations where mutation H1069Q accounts for 50% of WD alleles or more. The text was submitted authors English.     

Genetic analysis of 55 northern Vietnamese patients with Wilson disease: seven novel mutations in <Emphasis Type="BoldItalic">ATP7B</Emphasis>

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. The gene responsible for WD was discovered in 1993 and is located on chromosome 13 at 13q14.3. It encodes a copper-specific transporting P-type ATPase. Early diagnosis can improve treatment outcome and decrease the rate of disability or even mortality. We used Sanger sequencing to identify mutation hot spots in 55 northern Vietnamese with a clinical diagnosis of WD. Mutations were screened and detected by direct DNA sequencing. A total of 26 different ATP7B gene mutations were identified, including seven novel mutations (five nonsense and two missense mutations). The most frequent mutations were p.Ser105Ter (24.55%), p.Arg778Leu (5.45%) and p.Thr850Ile (4.55%). Mutation detection rate in exon 2 was 34.55% and ranked first, followed by exon 8 with 16.36%, and exon 18 with 10.91% each, thus, exons 2, 8 and 18 are the mutation hot spots for northern Vietnamese WD patients. These findings were different from previous studies in Asia. Our research established a suitable strategy for ATP7B gene testing in northern Vietnamese WD patients.     

A structural model of the copper ATPase ATP7B to facilitate analysis of Wilson disease-causing mutations and studies of the transport mechanism

The copper-transporting ATPase ATP7B has an essential role in human physiology, particularly for the liver and brain function. Inactivation of ATP7B is associated with a severe hepato-neurologic disorder, Wilson disease (WD). Hundreds of WD related mutations have been identified in ATP7B to date. The low frequency and the compound-heterozygous nature of causative mutations complicate the analysis of individual mutants and the establishment of genotype-phenotype correlations. To facilitate studies of disease-causing mutations and mechanistic understanding of WD, we have homology-modelled the ATP7B core (residues 643-1377) using the recent structure of the bacterial copper-ATPase LCopA as a template. The model, supported by evolutionary conservation and hydrophobicity analysis, as well as existing and new mutagenesis data, allows molecular interpretations of experimentally characterized clinical mutations. We also illustrate that structure and conservation can be used to grade potential deleterious effects for many WD mutations, which were clinically detected but have not yet been experimentally characterized. Finally, we compare the structural features of ATP7B and LCopA and discuss specific features of the eukaryotic copper pump.     

Tissue localization of the copper chaperone ATOX1 and its potential role in disease

ATOX1 is a cytoplasmic copper chaperone that interacts with the copper-binding domain of the membrane copper transporters ATP7A and ATP7B. ATOX1 has also been suggested to have a potential anti-oxidant activity. This study investigates the tissue-specific localization of the mouse homolog, Atox1, in mouse liver and kidney. Immunohistochemical studies in the liver localize the copper chaperone to hepatocytes surrounding both hepatic and central veins. In the kidney, Atox1 is localized to the cortex and the medulla. Cortex immunostaining is specific to glomeruli in both the juxtamedullary and cortical nephrons. Expression in the medulla appears to be associated with the loops of Henle. These data suggest that localized regions in the liver and kidney express Atox1 and have a role in copper homeostasis and/or anti-oxidant protection. Twenty-seven patients with Wilson disease-like phenotypes and two patients with Menkes disease-like phenotypes were screened for ATOX1 mutations with no alterations detected. The human phenotype resulting from mutations in ATOX1 remains unidentified.     

Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells

The combination of disease-specific human induced pluripotent stem cells (iPSC) and directed cell differentiation offers an ideal platform for modeling and studying many inherited human diseases. Wilson’s disease (WD) is a monogenic disorder of toxic copper accumulation caused by pathologic mutations of the ATP7B gene. WD affects multiple organs with primary manifestations in the liver and central nervous system (CNS). In order to better investigate the cellular pathogenesis of WD and to develop novel therapies against various WD syndromes, we sought to establish a comprehensive platform to differentiate WD patient iPSC into both hepatic and neural lineages. Here we report the generation of patient iPSC bearing a Caucasian population hotspot mutation of ATP7B. Combining with directed cell differentiation strategies, we successfully differentiated WD iPSC into hepatocyte-like cells, neural stem cells and neurons. Gene expression analysis and cDNA sequencing confirmed the expression of the mutant ATP7B gene in all differentiated cells. Hence we established a platform for studying both hepatic and neural abnormalities of WD, which may provide a new tool for tissue-specific disease modeling and drug screening in the future.     

Copper toxicosis in non-COMMD1 Bedlington terriers is associated with metal transport gene ABCA12

Wilson’s disease, caused by a mutation in the ATP-ase 7B gene, is the only genetically characterised human disease with inhibition of biliary copper excretion and toxic copper accumulation in liver and occasionally brain. A similar copper toxicosis occurs in Bedlington terriers (CT) with liver damage only. Although CT has been associated with a defect in the COMMD1 gene (COMMD1 ^del/del), Bedlington terriers with CT and lacking this mutation are also recognised (non-COMMD1 ^del/del).A study was designed to identify any other gene polymorphisms associated with copper toxicity in Bedlington terriers employing genome wide association studies (GWAS) followed by deep sequencing of the candidate region. Blood for DNA analysis and liver for confirmation of the diagnosis was obtained from 30 non-COMMD1 ^del/del Bedlington terriers comprising equal numbers of CT-affected dogs and controls. DNA was initially subjected to GWAS screening and then further sequencing to target the putative mutant gene.The study has identified a significant disease association with a region on chromosome 37 containing identified SNP’s which are highly significantly associated with non-COMMD1 ^del/del Bedlington terrier CT. This region contains the ABCA12 gene which bears a close functional relationship to ATP-ase 7B responsible for Wilson’s disease in man.     

Detection of His1069Gln mutation in Wilson disease by bidirectional PCR amplification of specific alleles (BI-PASA) test

Wilson disease (WD) is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase, ATP7B. The majority of known mutations affecting this gene are frequent in different populations, which may help to introduce rapid diagnostic procedures based on direct DNA analysis into routine clinical practise. The His1069Gln mutation in exon 14 is the most frequent one, accounting for 30-60% of all mutations in Caucasian patients. The aim of the present work was to introduce DNA-based direct analysis into routine molecular screening for the above mutation in Slovak WD patients and to assess its frequency in patients as well as in a control population. Twenty seven clinicaly diagnosed patients from twenty five families, twenty relatives of index patients and three hundred and six control DNA samples were tested using two different DNA-based methods: the earlier described amplification created restriction site (ACRS) for Alw21I in combination with nested PCR and the amplification refractory mutation system (ARMS). In 18 of 25 unrelated patients (72%), the mentioned genetic defect was present in at least one copy. In ten of them (40%), the above mutation was detected in homozygous and in eight individuals (32%) in heterozygous state. In seven WD patients (28%), this mutation was not detected. The allele frequency of His1069Gln in Slovak patients with WD was 56%, which was higher as reported in other populations. In a control group of 306 random DNA samples (612 alleles), the His1069Gln mutation was observed in 3 samples (carrier frequency 1%; allele frequency 0.49%). These frequencies correspond to figures observed in different population of European origin. Taken together, we have provided further evidence that the His1069Gln mutation is the prevalent ATP7B mutation in central-european WD patients. Although both methods used in this study worked in our hands reliably, there are in every-day use some drawbacks and limitations inherent to them (PCR reactions in two tubes, possibility of star activity or not complet digestion by restriction endonuclease, etc.). Therefore we developed a simpler, cost effective and rapid DNA diagnostic test based on bidirectional amplification of specific alleles (BI-PASA), which enables detection of homozygotes (wild and mutant) and heterozygotes, respectivelly, in one PCR reaction. The test was highly sensitive and specific, yielding no false-positive or false-negative results. Its reliability and discriminating power was tested on samples of 27 WD patients and 120 random control DNA's, previously genotyped by above mentioned methods. Comparing results of BI-PASA with ACRS and ARMS tests showed 100% concordance.     

Delineation of the spectrum of Wilson disease mutations in the Greek population and the identification of six novel mutations

In this study, we report the further results of an ongoing project on the delineation of the spectrum of mutations on the ATP7B gene in Wilson disease (WD) patients of Greek origin. We have analyzed 24 additional families and detected 16 mutations (five frameshifts, two splice site, two nonsense, and seven missense), of which six are novel. On adding these results to the ones already published by us, we conclude that WD shows a marked allelic heterogeneity in the Greek population. Of the total number of mutations so far detected, the most common eight account for the molecular defect in 72.8% of the WD chromosomes. The most frequent mutation is the His0169Gln, which has a frequency of 28.5%, similar to those reported in North European populations. Using these data, an efficient strategy of mutation screening for WD is possible in this population, thus improving the possibility of preclinical diagnosis.     

Late neurological presentations of Wilson disease patients in French population and identification of 8 novel mutations in the ATP7B gene.

Wilson disease (WD) is an autosomal recessive disorder of copper biliary excretion caused by an impaired function of ATP7B, a metal-transporting P-type ATPase encoded by WD gene. It results in copper accumulation, mostly in liver and brain tissues. Mutation analysis was carried out on 11 WD French unrelated patients presenting a predominant neurological form of this illness. SSCP and dHPLC analysis followed by sequencing of the 21 exons and their flanking introns were performed. Thirteen different mutations in a total of 17, and, among them, 10 novel variants were evidenced. Two deletions (c.654_655delCC and c.1745_1746delTA), 4 missense mutations (p.F763Y, p.G843R, p.D918A and p.L979Q), 1 nonsense mutation (p.Q1200X), 1 splice site mutation (c.1947-1G>C) and 2 intronic silent substitutions (c.2448-25G>T and c.3412+13T>A) were detected. These data extend the mutational spectrum of the disease, already known to be a very heterogeneous genetic disorder. As compared to hepatic manifestations, the phenotypes associated to these mutations confirm that neurological presentations associated with other mutations than p.H1069Q are also often late in their onset. Most of these neurological forms probably correspond to an attenuated impairment of copper metabolism, as compared to hepatic forms of the disease, mostly diagnosed earlier.