A serine-to-proline mutation in the copper-transporting P-type ATPase gene of the macular mouse

We have investigated the cDNA sequence of the copper-transporting P-type ATPase (Atp7a) gene of the macular mouse, a model for human Menkes disease. A point mutation (T to C) that results in substitution of proline for serine in a putative eighth transmembrane domain of the ATP7A was identified. This contrasts with abnormalities identified in the Atp7a of other mottled mouse strains: lack of expression of Atp7a mRNA in the dappled mouse, and a splicing mutation in the blotchy mouse. Received: 1 December 1996 / Accepted: 20 January 1997     

Rapid and robust screening of the Menkes disease/occipital horn syndrome gene

Menkes disease and occipital horn syndrome (OHS) are allelic neurogenetic disorders of copper transport associated with mutations in an X-linked gene, ATP7A. This gene encodes a copper-transporting P-type ATPase. The spectrum of mutations at the Menkes/OHS locus is estimated to include 1% chromosomal rearrangements and 15-20% large deletions, with the remaining defects involving small alterations. There is a compelling need for a rapid and reliable molecular diagnostic approach for patients and families impacted by these conditions. In addition to testing suspected affected males, carrier screening of females in Menkes/OHS families and prenatal evaluation of at-risk pregnancies will be enhanced by the wider availability of robust mutation analysis for this large (23-exon) locus. Here we describe a stepwise approach to mutation screening for these disorders that successfully identified molecular alterations in over 95% of our patient population (n = 49). This genomic DNA-based technique employs multiplex PCR, heteroduplex analysis, and direct sequencing, in a serial fashion. This approach should find application in molecular diagnostic laboratories in the United States and other countries. Currently, only a single European center provides commercial testing for unknown mutations in Menkes/OHS patients, even though these disorders occur worldwide.     

A comparison of phenotype and copper distribution in blotchy and brindled mutant mice and in nutritionally copper deficient controls

The murine mottled mutants brindled, Mo br, and blotchy, Mo blo, are valuable animal models for the study of mammalian copper metabolism. In this paper, we present data showing that a nutritionally copper deficient suckling mouse, Cu-, with strong phenotypic similarities to the brindled mutant can be produced by feeding genetically normal dams a copper deficient diet (0.1-0.4 ppm Cu2+) from the day of mating. Comparisons of copper distribution between the Cu- mice and brindled mutants indicate that when a small dose of copper (0.5-0.9 micrograms Cu2+) was administered by intracardiac injection, the copper was abnormally distributed, and that the pattern of tissue distribution was very similar in Cu- mice and brindled mutants 24 h after injection. When, however, a treatment dose (50 micrograms Cu2+) was injected subcutaneously, and tissues assayed 3 d after injection, copper distribution in Cu- mice and brindled mutants was clearly different. Copper deficiency in Cu- suckling mice is entirely derived from maternal effects. Evidence that maternal effects may also influence the survival and phenotype of the brindled and blotchy mutants was obtained by comparing the viability of mutants born to dams carrying mottled mutations on one or both X chromosomes.     

Localization of the translocation breakpoint in a female with Menkes syndrome to Xq13.2-q13.3 proximal to PGK-1.

Menkes syndrome is a rare X-linked recessive disorder characterized by an inability to metabolize copper. A female patient with both this disease and an X; autosome translocation with karyotype 46,X,t(X;2)(q13;q32.2) has previously been described. The translocation breakpoint in Xq13 coincides with a previous assignment of the Menkes gene at Xq13 by linkage data in humans and by analogy to the mottled mutations which are models for Menkes disease in the mouse. Therefore, this translocation probably interrupts the gene for Menkes syndrome in band Xq13. We describe here experiments to precisely map the translocation breakpoint within this chromosomal band. We have established a lymphoblastoid cell line from this patient and have used it to isolate the der(2) translocation chromosome (2pter----2q32::Xq13----Xqter) in human/hamster somatic cell hybrids. Southern blot analyses using a number of probes specific for chromosomes X and 2 have been studied to define precisely the location of the translocation breakpoint. Our results show that the breakpoint in this patient--and, therefore, likely the Menkes gene--maps to a small subregion of band Xq13.2-q13.3 proximal to the PGK1 locus and distal to all other Xq13 loci tested.     

Molecular diagnosis of Menkes disease: Genotype–phenotype correlation

Menkes syndrome is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene, encoding a copper-transporting P1B-type ATPase. To date, a total of approximately 160 different mutations have been reported worldwide. The clinical phenotypes observed in these patients include progressive neuro-degeneration, connective-tissue abnormalities and peculiar hair. There is phenotypic variability. While the majority of the patients do not survive early childhood, milder cases leading to longer survival have been reported. In this review we focus on mutations, identified in patients with milder forms of Menkes disease, and discuss the possibility of establishing a genotype–phenotype correlation. The presence of small amounts of normal protein, or the presence of partly functional protein variants containing a less essential amino acid substitution or a truncation of the N- or C-terminus, might all result in a milder, atypical phenotype. A clear phenotype–genotype correlation is however difficult to establish, clearly illustrated by the presence of inter- and even intra-familial variability.     

Intragenic deletions at Atp7a in mouse models for Menkes disease

Mottled mice have mutations in the copper-transporting ATPase Atp7a. They are proven models for the human disorder Menkes disease (MD), which results from mutations in a homologous gene. Mottled mice can be divided into three classes: class 1, in which affected males die before birth; class 2, in which affected males die in the early postnatal period; and class 3, in which affected males survive to adulthood. In humans, it has been shown that mutations that lead to a complete absence of functional protein cause classical MD, which is characterized by death of boys in early childhood. We hypothesized that the most severely affected mottled alleles would be the most likely to carry mutations equivalent to those causing classical MD and therefore undertook mutational analysis of several class 1 mottled alleles to assess whether these were appropriate models for the disease at the molecular level. Two novel mutations, a deletion of exons 11-14 in mottled spot and an insertion in exon 10 leading to missplicing in mottled candy, were identified. However, these are both "in-frame" mutations, as are the other eight Atp7a mutations reported to date, and therefore no frameshift or nonsense mutations have yet been associated with the mottled phenotype. This contrasts with the mutation spectrum associated with MD, emphasizing the need for caution when mottled mice are used as models for the clinical disorder.     

Genomic organization of ATOX1, a human copper chaperone

Background

Copper is an essential trace element that plays a critical role in the survival of all living organisms. Menkes disease and occipital horn syndrome (OHS) are allelic disorders of copper transport caused by defects in a X-linked gene (ATP7A) that encodes a P-type ATPase that transports copper across cellular membranes, including the trans-Golgi network. Genetic studies in yeast recently revealed a new family of cytoplasmic proteins called copper chaperones which bind copper ions and deliver them to specific cellular pathways. Biochemical studies of the human homolog of one copper chaperone, ATOX1, indicate direct interaction with the Menkes/OHS protein. Although no disease-associated mutations have been reported in ATOX1, mice with disruption of the ATOX1 locus demonstrate perinatal mortality similar to that observed in the brindled mice (Mo^br), a mouse model of Menkes disease. The cDNA sequence for ATOX1 is known, and the genomic organization has not been reported.

Results

We determined the genomic structure of ATOX1. The gene contains 4 exons spanning a genomic distance of approximately 16 kb. The translation start codon is located in the 3' end of exon 1 and the termination codon in exon 3. We developed a PCR-based assay to amplify the coding regions and splice junctions from genomic DNA. We screened for ATOX1 mutations in two patients with classical Menkes disease phenotypes and one individual with occipital horn syndrome who had no alterations detected in ATP7A, as well as an adult female with chronic anemia, low serum copper and evidence of mild dopamine-beta-hydroxylase deficiency and no alterations in the ATOX1 coding or splice junction sequences were found.

Conclusions

In this study, we characterized the genomic structure of the human copper chaperone ATOX1 to facilitate screening of this gene from genomic DNA in patients whose clinical or biochemical phenotypes suggest impaired copper transport.

     

Mutation in the CPC motif-containing 6th transmembrane domain affects intracellular localization, trafficking and copper transport efficiency of ATP7A protein in mosaic mutant mice--an animal model of Menkes disease

Copper is an essential micronutrient for all living organisms. ATP7A protein is a copper-transporting ATPase which plays a vital role in the maintenance of cellular copper homeostasis in mammals. This protein is retained within the trans-Golgi network, but after binding copper it can be translocated to the cell membrane to participate in the efflux of excess Cu. Mutation of the ATP7A gene in humans results in the severe neurodegenerative disorder, Menkes disease. The mouse ATP7A homolog encodes a protein that plays the same role in copper transport. Mosaic mutant mice display a lethal phenotype which resembles Menkes disease, although the underlying molecular defect has not been characterized until now. In the present study we identified a G to C nucleotide exchange in exon 15 of the Atp7a gene in mosaic mutants, which resulted in an arginine to proline substitution in the highly conserved 6th transmembrane domain of the ATP7A protein. This mutated protein was mislocalized in kidney cells isolated from mosaic mutant mice, and following exposure of these cells to increased copper concentrations it was not translocated to the plasma membrane. Disturbance of ATP7A function in mosaic mice results in increased copper accumulation in the small intestine and kidneys, and in Cu deficiency in the brain, liver and heart. Mouse models of Menkes disease belong to the mottled mutant group. The mosaic mutant represents another interesting animal model for Menkes disease that will be of value in research on copper metabolism and transport in mammals.     

Correction of a mouse model of Menkes disease by the human Menkes gene

The brindled mouse is an accurate model of the fatal human X-linked copper deficiency disorder, Menkes disease. Males carrying the mutant allele of the Menkes gene orthologue Atp7a die in the second week of life. To determine whether the genetic defect in the brindled mice could be corrected by expression of the human Menkes gene, male transgenic mice expressing ATP7A from the chicken beta-actin composite promoter (CAG) were mated with female carriers of the brindled mutation (Atp7a(Mo-br)). Mutant males carrying the transgene survived and were fertile but the copper defect was not completely corrected. Unexpectedly males corrected with one transgenic line (T25#5) were mottled and resembled carrier females, this effect appeared to be caused by mosaic expression of the transgene. In contrast, males corrected with another line (T22#2) had agouti coats. Copper concentrations in tissues of the rescued mutants also resembled those of the heterozygous females, with high levels in kidney (84.6+/-4.9 microg/g in corrected males vs. 137.0+/-44.3 microg/g in heterozygotes) and small intestine (15.6+/-2.5 microg/g in corrected males vs. 15.7+/-2.8 microg/g in heterozygotes). The results show that the Menkes defect in mice is corrected by the human Menkes gene and that adequate correction is obtained even when the transgene expression does not match that of the endogenous gene.     

Phenotypic and genetic characterization of the Atp7a(Mo-Tohm) mottled mouse: a new murine model of Menkes disease

Mottled Tohoku (Atp7a(Mo-Tohm) or Mo(Tohm)) is an X-linked mutation with mottled pigmentation in heterozygous (Mo(Tohm)/+) females and is embryonic lethal at E11 in hemizygous (Mo(Tohm)/Y) males. Copper levels were low in the brain and high in the intestine of Mo(Tohm) mice. Two congenic strains with ICR or C57BL/6 (B6) background were produced for genetic and phenotypic analyses and revealed that Mo(Tohm)/+ females with ICR background survived until adulthood, while most with B6 background died within 2 days after birth. The Mo(Tohm)/Y males with both backgrounds died at around E11. Massive hemorrhage was shown in the yolk sac cavity with irregular attachment between the mesoderm and the endothelial cells of blood vessels in the embryos at E10.5, suggesting that this irregular attachment causes embryonic lethality. The Mo(Tohm) mutant had a 1440-bp deletion between intron 22 and exon 23 of the Atp7a gene. Mo(Tohm)/Y males with the wild-type Atp7a cDNA transgene were rescued from embryonic lethality, confirming that the Mo(Tohm) mutant is caused by the defect in the Atp7a gene. This mutant mouse is the most severe model of human Menkes disease in mottled mice established to date and one of the useful models for understanding the gene function of Menkes disease.     

Expression of the human Menkes ATPase in Xenopus laevis oocytes

Menkes disease is an X-linked disorder of copper metabolism that is usually fatal. The affected gene has recently been cloned and encodes one of the two human copper ATPases. If the Menkes ATPase is defective, copper is trapped in the intestinal mucosa, leading to systemic copper deficiency. In order to study copper transport by this ATPase and the effects of disease mutations on its function, we developed a Xenopus laevis oocyte expression system. Wild-type Menkes ATPase cDNA and a fusion of this gene with the green fluorescent protein (GFP) gene was transcribed in vitro and the mRNA injected into oocytes. Expression in oocytes was analyzed by Western blotting and fluorescence microscopy. The Menkes ATPase-GFP chimera appeared to localize primarily to the plasma membrane as assessed by confocal microscopy. This system should thus provide an interesting new tool to study the function of the Menkes ATPase.     

Menkes copper-translocating P-type ATPase (ATP7A): biochemical and cell biology properties,and role in Menkes disease

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a ubiquitous protein that regulates the absorption of copper in the gastrointestinal tract. Inside cells the protein has a dual function: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper. The latter property is achieved through copper-dependent vesicular trafficking of the Menkes protein to the plasma membrane of the cell. The trafficking mechanism and catalytic activity combine to facilitate absorption and intercellular transport of copper. The mechanism of catalysis and copper-dependent trafficking of the Menkes protein are the subjects of this review. Menkes disease, a systemic copper deficiency disorder, is caused by mutations in the gene encoding the Menkes protein. The effect of these mutations on the catalytic cycle and the cell biology of the Menkes protein, as well as predictions of the effect of particular mutant MNKs on observed Menkes disease symptoms will also be discussed.     

Structure of the actuator domain from the Archaeoglobus fulgidus Cu(+)-ATPase

Copper homeostasis is maintained in part by membrane-bound P(1B)-type ATPases that are found in all organisms and drive the transport of this essential, yet toxic, metal ion across cellular membranes. CopA from Archaeoglobus fulgidus is a hyperthermophilic member of this ATPase subfamily and is homologous to the human Wilson and Menkes disease ATPases. To gain insight into Cu(+)-ATPase function, the structure of the CopA actuator domain (A-domain) was determined to 1.65 A resolution. The CopA A-domain functions to couple ATP hydrolysis in the ATP binding domain (ATPBD) with structural rearrangements of critical transmembrane segments. Its fold is quite similar to that of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) A-domain, with the exception of an external loop region. On the basis of sequence and structural comparisons, specific residues that probably interact with the CopA ATPBD have been identified. Comparisons to the Wilson and Menkes disease A-domains reveal the presence of an additional loop that may be associated with regulatory functions in eukaryotic Cu(+)-ATPases. Finally, several mutations in the Wilson and Menkes disease ATPases occur in the A-domain, and their likely effects on function can be inferred from the CopA A-domain structure.     

Copper-regulated trafficking of the Menkes disease copper ATPase is associated with formation of a phosphorylated catalytic intermediate

The Menkes protein (MNK; ATP7A) is a copper-transporting P-type ATPase that is defective in the copper deficiency disorder, Menkes disease. MNK is localized in the trans-Golgi network and transports copper to enzymes synthesized within secretory compartments. However, in cells exposed to excessive copper, MNK traffics to the plasma membrane where it functions in copper efflux. A conserved feature of all P-type ATPases is the formation of an acyl-phosphate intermediate, which occurs as part of the catalytic cycle during cation transport. In this study we investigated the effect of mutations within conserved catalytic regions of MNK on intracellular localization and trafficking from the trans-Golgi network (TGN). Our findings suggest that mutations that block formation of the phosphorylated catalytic intermediate also prevent copper-induced relocalization of MNK from the TGN. Furthermore, mutations in the phosphatase domain, which resulted in hyperphosphorylation of MNK, caused constitutive trafficking from the TGN to the plasma membrane. A similar effect on trafficking was observed with a phosphatase mutation in the closely related copper ATPase, ATP7B, affected in Wilson disease. These findings suggest that the copper-induced trafficking of the Menkes and Wilson disease copper ATPases is associated with the phosphorylated intermediate that is formed during the catalysis of these pumps. Our findings describe a novel mechanism for regulating the subcellular location of a transport protein involving the recognition of intermediate conformations during catalysis.     

Missense Mutations in the Copper Transporter Gene ATP7A Cause X-Linked Distal Hereditary Motor Neuropathy

Distal hereditary motor neuropathies comprise a clinically and genetically heterogeneous group of disorders. We recently mapped an X-linked form of this condition to chromosome Xq13.1-q21 in two large unrelated families. The region of genetic linkage included ATP7A, which encodes a copper-transporting P-type ATPase mutated in patients with Menkes disease, a severe infantile-onset neurodegenerative condition. We identified two unique ATP7A missense mutations (p.P1386S and p.T994I) in males with distal motor neuropathy in two families. These molecular alterations impact highly conserved amino acids in the carboxyl half of ATP7A and do not directly involve the copper transporter''s known critical functional domains. Studies of p.P1386S revealed normal ATP7A mRNA and protein levels, a defect in ATP7A trafficking, and partial rescue of a S. cerevisiae copper transport knockout. Although ATP7A mutations are typically associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome, we demonstrate here that certain missense mutations at this locus can cause a syndrome restricted to progressive distal motor neuropathy without overt signs of systemic copper deficiency. This previously unrecognized genotype-phenotype correlation suggests an important role of the ATP7A copper transporter in motor-neuron maintenance and function.     

Function and Regulation of the Mammalian Copper-transporting ATPases: Insights from Biochemical and Cell Biological Approaches

Copper is an essential trace element that plays a very important role in cell physiology. In humans, disruption of normal copper homeostasis leads to severe disorders, such as Menkes disease and Wilson's disease. Recent genetic, cell biological, and biochemical studies have begun to dissect the molecular mechanisms involved in transmembrane transport and intracellular distribution of copper in mammalian cells. In this review, we summarize the advances that have been made in understanding of structure, function, and regulation of the key human copper transporters, the Menkes disease and Wilson's disease proteins.     

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.     

Essential roles in development and pigmentation for the Drosophila copper transporter DmATP7

Defects in the mammalian Menkes and Wilson copper transporting P-type ATPases cause severe copper homeostasis disease phenotypes in humans. Here, we find that DmATP7, the sole Drosophila orthologue of the Menkes and Wilson genes, is vital for uptake of copper in vivo. Analysis of a DmATP7 loss-of-function allele shows that DmATP7 is essential in embryogenesis, early larval development, and adult pigmentation and is probably required for copper uptake from the diet. These phenotypes are analogous to those caused by mutation in the mouse and human Menkes genes, suggesting that like Menkes, DmATP7 plays at least two roles at the cellular level: delivering copper to cuproenzymes required for pigmentation and neuronal function and removing excess cellular copper via facilitated efflux. DmATP7 displays a dynamic and unexpected expression pattern in the developing embryo, implying novel functions for this copper pump and the lethality observed in DmATP7 mutant flies is the earliest seen for any copper homeostasis gene.