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Graphical abstract, ATP7A is a critical copper transporter involved in Menkes Disease, Occipital horn Syndrome and X-linked distal spinal muscular atrophy type 3 which are X linked genetic disorders. These are rare diseases and their genetic epidemiology of the diseases is unknown. A number of genetic variants in the genes have been reported in published literature as well as databases, however, understanding the pathogenicity of variants and genetic epidemiology requires the data to be compiled in a unified format. To this end, we systematically compiled genetic variants from published literature and datasets. Each of the variants were systematically evaluated for evidences with respect to their pathogenicity and classified as per the American College of Medical Genetics and the Association of Molecular Pathologists (ACMG-AMP) guidelines into Pathogenic, Likely Pathogenic, Benign, Likely Benign and Variants of Uncertain Significance. Additional integrative analysis of population genomic datasets provides insights into the genetic epidemiology of the disease through estimation of carrier frequencies in global populations. To deliver a mechanistic explanation for the pathogenicity of selected variants, we also performed molecular modeling studies. Our modeling studies concluded that the small structural distortions observed in the local structures of the protein may lead to the destabilization of the global structure. To the best of our knowledge, ATP7A Clinical Genetics Resource is one of the most comprehensive compendium of variants in the gene providing clinically relevant annotations in gene.

Menkes disease (MD) is a rare and often lethal X-linked recessive syndrome, characterized by generalized alterations in copper transport and metabolism, linked to mutations in the ATPase copper transporting α (ATP7A) gene. Our objective was to identify genomic alterations and circulating proteomic profiles related to MD assessing their potential roles in the clinical features of the disease. We describe the case of a male patient of 8 months of age with silvery hair, tan skin color, hypotonia, alterations in neurodevelopment, presence of seizures, and low values of plasma ceruloplasmin. Trio-whole-exome sequencing (Trio-WES) analysis, plasma proteome screening, and blood cell migration assays were carried out. Trio-WES revealed a hemizygous change c.4190C &gt, T (p.S1397F) in exon 22 of the ATP7A gene. Compared with his parents and with child controls, 11 plasma proteins were upregulated and 59 downregulated in the patient. According to their biological processes, 42 (71.2%) of downregulated proteins had a participation in cellular transport. The immune system process was represented by 35 (59.3%) downregulated proteins (p = 9.44 × 10–11). Additional studies are necessary to validate these findings as hallmarks of MD.

Background Menkes disease is a congenital neurodegenerative disorder caused by ATP7A gene mutations. Clinical features include epilepsy, growth delay, reduced muscle strength, skin laxity, abnormal hair, and urologic abnormalities. Case presentation We describe an infant with developmental delay, neurologic degeneration, and kinky hair. Molecular test revealed a novel heterozygous mutation in exon 21 of the ATP7A gene. The genotype and phenotype of the patient were compared with those of the patients reported in the literature. Conclusion We propose that this mutation caused a dysfunctional protein resulting in classical Menkes disease. This case adds to the spectrum of pathogenic variants of the ATP7A gene known to cause disease.

CRISPR/Cas9 is a powerful tool for producing genomic insertions and deletions (indels) to interrogate gene function. Modified CRISPR/Cas9 protocols can produce targeted genetic changes that are more precise than indels, but founder recovery is less efficient. Focusing on producing missense mutations in zebrafish using single-stranded oligo deoxynucleotide (ssODN) donor templates, we pioneered a strategy of adding synonymous changes to create novel restriction-enzyme (RE) sites, allowing detection of rare precise edits in a modified fluorescent-PCR fragment assay. We have named this process TIARS (test for incorporation of added recognition sites). Aided by TIARS, we induced two distinct amino-acid substitutions (T979I and P1387S) in the atp7a gene among somatic tissues of CRISPR-Cas9-treated F0 zebrafish. One of these F0s transmitted the allele to atp7aT979I/+ F1 progeny, and trans-heterozygosity of this allele against a null atp7a allele causes hypopigmentation, consistent with more severe pigment deficits in zebrafish or humans carrying only null mutations in atp7a/ATP7A. Design of ssODNs with novel RE recognition sites is labor-intensive, so we developed an in silico tool, TIARS Designer, and performed bioinformatic validation indicating that TIARS should be generalizable to other genes and experimental systems that employ donor template DNA.

Background: The ATP7A gene encodes a copper transporter whose mutations cause Menkes disease, occipital horn syndrome (OHS), and, less frequently, ATP7A-related distal hereditary motor neuropathy (dHMN). Here we describe a family with OHS caused by a novel mutation in the ATP7A gene, including a patient with a comorbid dHMN that worsened markedly after being treated with copper histidinate. Methods: We studied in detail the clinical features of the patients and performed a genomic analysis by using TruSight One Expanded Sequencing Panel. Subsequently, we determined the ATP7A and ATP7B expression levels, mitochondrial membrane potential, and redox balance in cultured fibroblasts of Patient 1. Results: We found a novel ATP7A late truncated mutation p.Lys1412AsnfsX15 in the two affected members of this family. The co-occurrence of OHS and dHMN in Patient 1 reveals the variable phenotypic expressivity of the variant. A severe clinical and neurophysiologic worsening was observed in the dHMN of Patient 1 when he was treated with copper replacement therapy, with a subsequent fast recovery after the copper histidinate was withdrawn. Functional studies revealed that the patient had low levels of both ATP7A and ATP7B, the other copper transporter, and high levels of superoxide ion in the mitochondria. Conclusions: Our findings broaden the clinical spectrum of ATP7A-related disorders and demonstrate that two clinical phenotypes can occur in the same patient. The copper-induced toxicity and low levels of both ATP7A and ATP7B in our patient suggest that copper accumulation in motor neurons is the path-ogenic mechanism in ATP7A-related dHMN. (c) 2021 Elsevier Inc. All rights reserved.

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.

Supervisor:塚原 俊文, 先端科学技術研究科, 博士

Mosaic mutant mice displaying functional dysfunction of Atp7a copper transporter (the Menkes ATPase) are an established animal model of Menkes disease and constitute a convenient tool for investigating connections between copper and iron metabolisms. This model allows to explore changes in iron metabolism in suckling mutant mice suffering from systemic copper deficiency as well as in young and adult ones undergone copper therapy, which reduces lethal effect of the Atp7a gene mutation. Our recent study demonstrated that 14-day-old mosaic mutant males display blood cell abnormalities associated with intravascular hemolysis, and show disturbances in the functioning of the hepcidin-ferroportin regulatory axis, which controls systemic iron homeostasis. We thus aimed to check whether copper supplementation recovers mutants from hemolytic insult and rebalance systemic iron regulation. Copper supplementation of 14-day-old mosaic mutants resulted in the reestablishment of hematological status, attenuation of hepicidin and concomitant induction of the iron exporter ferroportin/Slc40a1 expression in the liver, down-regulated in untreated mutants. Interestingly, treatment of wild-type males with copper, induced hepcidin-independent up-regulation of ferroportin protein level in hepatic macrophages in both young and adult (6-month-old) animals. Stimulatory effect of copper on ferroportin mRNA and protein levels was confirmed in bone marrow-derived macrophages isolated from both wild-type and mosaic mutant males. Our study indicates that copper is an important player in the regulation of the Slc40a1 gene expression.

ATP7A encodes a copper-transporting P-type ATPase and is one of 23 genes in which mutations produce distal hereditary motor neuropathy (dHMN), a group of diseases characterized by length-dependent axonal degeneration of motor neurons. We have generated induced pluripotent stem cell (iPSC)-derived motor neurons from a patient with the p.T994I ATP7A gene mutation as an in vitro model for X-linked dHMN (dHMNX). Patient motor neurons show a marked reduction of ATP7A protein levels in the soma when compared to control motor neurons and failed to upregulate expression of ATP7A under copper-loading conditions. These results recapitulate previous findings obtained in dHMNX patient fibroblasts and in primary cells from a rodent model of dHMNX, indicating that patient iPSC-derived motor neurons will be an important resource for studying the role of copper in the pathogenic processes that lead to axonal degeneration in dHMNX., Summary: The authors describe a neuronal model to investigate how mutations in the copper transporter ATP7A cause axonal degeneration in the peripheral nervous system.

Occipital horn syndrome (OHS) is a rare connective tissue disorder caused by pathogenic variants in ATP7A, encoding a copper transporter. The main clinical features, including cutis laxa, bony exostoses, and bladder diverticula are attributed to a decreased activity of lysyl oxidase (LOX), a cupro-enzyme involved in collagen crosslinking. The absence of large case series and natural history studies precludes efficient diagnosis and management of OHS patients. This study describes the clinical and molecular characteristics of two new patients and 32 patients previously reported in the literature. We report on the need for long-term specialized care and follow-up, in which MR angiography, echocardiography and spirometry should be incorporated into standard follow-up guidelines for OHS patients, next to neurodevelopmental, orthopedic and urological follow-up. Furthermore, we report on ultrastructural abnormalities including increased collagen diameter, mild elastic fiber abnormalities and multiple autophagolysosomes reflecting the role of lysyl oxidase and defective ATP7A trafficking as pathomechanisms of OHS.

Amyotrophic lateral sclerosis (ALS) is a severe disease causing motor neuron death, but a complete cure has not been developed and related genes have not been defined in more than 80% of cases. Here we compared whole genome sequencing results from a male ALS patient and his healthy parents to identify relevant variants, and chose one variant in the X-linked ATP7A gene, M1311V, as a strong disease-linked candidate after profound examination. Although this variant is not rare in the Ashkenazi Jewish population according to results in the genome aggregation database (gnomAD), CRISPR-mediated gene correction of this mutation in patient-derived and re-differentiated motor neurons drastically rescued neuronal activities and functions. These results suggest that the ATP7A M1311V mutation has a potential responsibility for ALS in this patient and might be a potential therapeutic target, revealed here by a personalized medicine strategy., Yeomin Yun, Sung-Ah Hong et al. compare the whole-genome sequence from a male with amyotrophic lateral sclerosis (ALS) to his healthy parents, identifying the M1311V variant in the X-lined ATP7A gene. They show that CRISPR-mediated gene correction in patient-derived neurons rescues neuronal activity.

Background Menkes disease (MD) is a rare X-linked hereditary multisystemic disorder that is caused by dysfunction of copper metabolism. Patients with MD typically present with progressive neurodegeneration, some connective tissue abnormalities, and characteristic “kinky” hair. In addition, various types of urological complications are frequent in MD because of underlying connective tissue abnormalities. In this study, we studied the clinical features and outcomes of MD, focusing on urological complications. Methods A total of 14 unrelated Korean pediatric patients (13 boys and 1 girl) with MD were recruited, and their phenotypes and genotypes were analyzed by retrospective review of their medical records. Results All the patients had early-onset neurological deficit, including developmental delay, seizures, and hypotonia. The girl patient showed normal serum copper and ceruloplasmin levels as well as milder symptoms. Mutational analysis of the ATP7A gene revealed 11 different mutations in 12 patients. Bladder diverticula was the most frequent urological complication: 8 (57.1%) in the 14 patients or 8 (72.7%) in the 11 patients who underwent urological evaluation. Urological imaging studies were performed essentially for the evaluation of accompanying urinary tract infections. Four patients had stage II chronic kidney disease at the last follow-up. Conclusion Urologic problems occurred frequently in MD, with bladder diverticula being the most common. Therefore, urological imaging studies and appropriate management of urological complications, which may prevent or reduce the development of urinary tract infections and renal parenchymal damage, are required in all patients with MD., Graphical Abstract

Menkes disease, also termed as “Menkes's syndrome”, is a disastrous infantile neurodegenerative disorder originated by diverse mutations in cupric cation-transport gene called ATP7A. This gene encodes a protein termed as copper transporting P-type ATP ase, essential for copper ion transport from intestine to the other parts of our body along with other transporters like copper transporter receptor 1 and divalent metal transporter 1. The copper transportation is vital in the neuronal development and synthesis of various enzymes. It is found to be an appreciated trace element for normal biological functioning but toxic in excess. It is essential for the metallation of cuproenzymes which is responsible for the biosynthesis of neurotransmitters and other vital physiological mechanisms. Copper is also actively involved in the transmission pathway of N-methyl-D-aspartate receptors and its subsequent molecular changes in neural cells. The expression of ATP7A gene in regions of brain depicts the importance of copper in neural development and stabilization. Studies revealed that the mutation of ATP7A gene leads the pathophysiology of various neurodegenerative disorders. This review focused on the normal physiological function of the gene with respect to their harmful outcome of the mutated gene and its associated deficiency which detriments the neural mechanism in Menkes patients.

Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7a LysMcre mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7a LysMcre mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7a LysMcre mice were also more susceptible to systemic infection by S . Typhimurium than wild-type mice. Deletion of the S . Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7a LysMcre mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S . Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection.

Menkes disease is caused by mutations in the ATP7A gene that lead to intracellular copper transport defects and characterized by brownish twisted...

Copper is an essential nutrient element required for normal development and physiology of live organisms; however, excess copper is harmful.1–7 Absorption, retention, and excretion of copper are tightly regulated to maintain copper homeostasis by a systemic balance of influx and efflux of copper ions under physiologic conditions.4,8,9 Wilson’s disease is caused by mutation of the ATP7B gene,10,11 which encodes a Cu+-transporting β polypeptide ATPase, and is characterized by hepatic and neurological disorders resulting from the toxicity of the accumulation of excess copper ions in the liver and brain tissues. In contrast, Menkes disease is caused by mutation of the ATP7A gene, which encodes a Cu+-transporting α polypeptide ATPase, and is characterized by neurological disorders resulting from cerebral copper deficiency due to reduced absorption of copper and subsequent reduction of copper influx to the brain.12–15 Significant progress has been made in describing the role of various copper transporters and chaperones in the regulation of copper metabolism at the molecular and cellular levels.16–20 However, many mechanistic details of the systemic regulation of copper metabolism remain elusive, partly due to the lack of a reliable tool for real-time kinetic analysis of copper fluxes in live organisms. Development of a tool for non-invasive measurement of copper fluxes in live organisms is not only significant for studying the systemic regulation of copper metabolism, but also significant for studying the pathophysiology of Wilson’s disease, Menkes disease, and other inherited and acquired human copper metabolism disorders.

Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene. Whereas most of the patients exhibit a severe classical form, about 9% of the patients exhibit a milder form of Menkes disease. The mildest form is called occipital horn syndrome (OHS). Mutations in the ATP7A gene can be identified in 95-98% of the Menkes disease patients by standard screening techniques. Investigation of RNA isolated from the fibroblasts of eleven patients with no identified mutations was performed, and revealed inclusion of new pseudo-exons into the ATP7A mRNA from three unrelated patients: two patients with OHS and one patient with classical Menkes disease. The pseudo-exons were inserted between exons 10 and 11, between exons 16 and 17 and between exons 14 and 15 in the three patients, as a result of deep intronic mutations. This is the first time the activation of pseudo-exons is demonstrated in the ATP7A gene, and it demonstrates the usefulness of RNA analysis, in terms of revealing disease-causing mutations in noncoding regions. The fact that three different mutations cause disease by the activation of pseudo-exon inclusion also indicates that in Menkes disease this is an important mechanism, which has hitherto been overlooked.

Menkes disease (MD) is a lethal multisystemic disorder of copper metabolism. Progressive neurodegeneration and connective tissue disturbances, together with the peculiar "kinky" hair, are the main manifestations. MD is inherited as an X-linked recessive trait, and as expected the vast majority of patients are males. MD occurs because of mutations in the ATP7A gene and the vast majority of ATP7A mutations are intragenic mutations or partial gene deletions. ATP7A is an energy-dependent transmembrane protein, which is involved in the delivery of copper to the secreted copper enzymes and in the export of surplus copper from cells. Severely affected MD patients die usually before the third year of life. A cure for the disease does not exist, but very early copper-histidine treatment may correct some of the neurological symptoms. This study reviews 274 published and 18 novel disease causing mutations identified in 370 unrelated MD patients, nonpathogenic variants of ATP7A, functional studies of the ATP7A mutations, and animal models of MD.

Copper balance is disturbed in two inborn errors: Wilson disease and Menkes disease. Wilson disease, or hepatolenticular degeneration, is caused by mutations in the ATP7B gene and is characterised by a gradual accumulation of copper in the liver and, secondarily, in other organs, such as brain, kidney and cornea. Clinical symptoms result from copper accumulation in the liver and/or the brain. Early treatment with copper chelators or zinc is generally effective. Menkes disease is an X-linked disorder due to mutations in the ATP7A gene. The disorder is characterised by a general copper deficiency. Patients manifest progressive neurodegeneration, which is usually fatal in infancy or childhood. Early therapy with copper histidine can be effective in selected patients. Occipital horn syndrome and a rare phenotype, X-linked distal hereditary motor neuropathy, are also due to ATP7A mutations and can be observed in older children or adults.

Menkes disease is an X-linked recessive lethal multisystemic disorder of copper metabolism. Progressive neurodegeneration, connective tissue disturbances, and peculiar kinky hair are the main manifestations. The low serum copper and ceruloplasmin suggests the diagnosis, which is confirmed by mutation analysis of the ATP7A gene. We report an exceptional presentation of classic Menkes disease with neonatal erythroderma. Genetic study revealed a deletion in exons 8 to 12 in the ATP7A gene. This study could allow pediatricians and pediatric dermatologists to diagnose the disorder as early as possible to establish prompt treatment with parenteral copper-histidine supplementation to improve prognosis.

Menkes disease is a multi-systemic copper metabolism disorder caused by mutations in the X-linked ATP7A gene and characterized by progressive neurodegeneration and severe connective tissue defects. The ATP7A protein is a copper (Cu)-transporting ATPase expressed in all tissues and plays a critical role in the maintenance of copper homeostasis in cells of the whole body. ATP7A participates in copper absorption in the small intestine and in copper transport to the central nervous system (CNS) across the blood-brain-barrier (BBB) and blood–cerebrospinal fluid barrier (BCSFB). Cu is essential for synaptogenesis and axonal development. In cells, ATP7A participates in the incorporation of copper into Cu-dependent enzymes during the course of its maturation in the secretory pathway. There is a high degree of homology (>80%) between the human ATP7A and murine Atp7a genes. Mice with mutations in the Atp7a gene, called mottled mutants, are well-established and excellent models of Menkes disease. Mottled mutants closely recapitulate the Menkes phenotype and are invaluable for studying Cu-metabolism. They provide useful models for exploring and testing new forms of therapy in Menkes disease. Recently, non-mammalian models of Menkes disease, Drosophila melanogaster and Danio rerio mutants were used in experiments which would be technically difficult to carry out in mammals.

Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment (daily subcutaneous copper injections) is not entirely effective in the majority of affected individuals. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice; 86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-β-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. Our findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease.

Copper is an essential trace metal for physiological functions, whereas copper overload causes cytotoxicity in living organisms. Genetically determined systems regulate acquisition, distribution and storage for copper maintenance and homeostasis. The Human ATP7A copper transport ATPase modulates intracellular copper distribution, which is critical for copper-dependent enzymes such as superoxide dismutase (SOD1). To investigate the role of zebrafish ATP7A in copper homeostasis, zebrafish atp7a gene expression was reduced for analysis of downstream cellular function. The results demonstrated that zebrafish sod1 has lower expression in atp7a-knockdown fish. Similarly, zebrafish sp1, a transcriptional regulator of sod1, also shows reduced expression in atp7a-knockdown fish. The lower expression of sod1 resulting from atp7a knockdown is independent to p53 gene activation. The knockdown of atp7a and copper chelator NeoC results in hypopigmentation and notochord deformation in zebrafish. Addition of exogenous copper alleviated the impaired development. Interestingly, both sod1 and sp1 transcripts are reduced in the presence of NeoC and increased with exogenous copper, suggesting that the expression of sod1 and sp1 are directly affected by copper status. This is the first report to demonstrate a hierarchic gene expression of copper homeostatic genes between atp7a, sp1 and sod1 in zebrafish.

This Review summarizes recent advances in understanding copper-transporting ATPase 1 (ATP7A), and examines the neurological phenotypes associated with dysfunction of this protein. Involvement of ATP7A in axonal outgrowth, synapse integrity and neuronal activation underscores the fundamental importance of copper metabolism to neurological function. Defects in ATP7A cause Menkes disease, an infantile-onset, lethal condition. Neonatal diagnosis and early treatment with copper injections enhance survival in patients with this disease, and can normalize clinical outcomes if mutant ATP7A molecules retain small amounts of residual activity. Gene replacement rescues a mouse model of Menkes disease, suggesting a potential therapeutic approach for patients with complete loss-of-function ATP7A mutations. Remarkably, a newly discovered ATP7A disorder—isolated distal motor neuropathy—has none of the characteristic clinical or biochemical abnormalities of Menkes disease or its milder allelic variant occipital horn syndrome (OHS), instead resembling Charcot–Marie–Tooth disease type 2. These findings indicate that ATP7A has a crucial but previously unappreciated role in motor neuron maintenance, and that the mechanism underlying ATP7A-related distal motor neuropathy is distinct from Menkes disease and OHS pathophysiology. Collectively, these insights refine our knowledge of the neurology of ATP7A-related copper transport diseases and pave the way for further progress in understanding ATP7A function.

Copper is a trace element that is essential for the normal growth and development of all living organisms. In mammals, the ATP7A Cu-transporting ATPase is a key protein that is required for the maintenance of copper homeostasis. In both humans and mice, the ATP7A protein is coded by the X-linked ATP7A/Atp7a gene. Disturbances in copper metabolism caused by mutations in the ATP7A/Atp7a gene lead to severe metabolic syndromes Menkes disease in humans and the lethal mottled phenotype in mice. Mosaic is one of numerous mottled mutations and may serve as a model for a severe Menkes disease variant. In Menkes patients, mutations in the ATP7A gene often result in a decreased level of the normal ATP7A protein. The aim of this study was to analyse the expression of the Atp7a gene in mosaic mutants in early postnatal development, a critical period for starting copper supplementation therapy in both Menkes patients and mutant mice. Using real-time quantitative RT-PCR, we analysed the expression of the Atp7a gene in the brain, kidney and liver of newborn (P0.5) and suckling (P14) mice. Our results indicate that in mosaic P0.5 mutants, the Atp7a mRNA level is decreased in all analysed organs in comparison with wild-type animals. In two week-old mutants, a significant decrease was observed only in the kidney. In contrast, their hepatic level of Atp7a tended to be higher than in wild-type mice. We speculate that disturbance in the expression of the Atp7a gene and, consequently, change in the copper concentration of the organs, may contribute to the early fatal outcome of mosaic males.

The trace metal copper is essential for a variety of biological processes, but extremely toxic when present in excessive amounts. Therefore, concentrations of this metal in the body are kept under tight control. Central regulators of cellular copper metabolism are the copper-transporting P-type ATPases ATP7A and ATP7B. Mutations in ATP7A or ATP7B disrupt the homeostatic copper balance, resulting in copper deficiency ( Menkes disease) or copper overload ( Wilson disease), respectively. ATP7A and ATP7B exert their functions in copper transport through a variety of interdependent mechanisms and regulatory events, including their catalytic ATPase activity, copper-induced trafficking, post-translational modifications and protein - protein interactions. This paper reviews the extensive efforts that have been undertaken over the past few years to dissect and characterise these mechanisms, and how these are affected in Menkes and Wilson disease. As both disorders are characterised by an extensive clinical heterogeneity, we will discus how the underlying genetic defects correlate with the molecular functions of ATP7A and ATP7B and with the clinical expression of these disorders.

The trace metal copper is essential for a variety of biological processes, but extremely toxic when present in excessive amounts. Therefore, concentrations of this metal in the body are kept under tight control. Central regulators of cellular copper metabolism are the copper-transporting P-type ATPases ATP7A and ATP7B. Mutations in ATP7A or ATP7B disrupt the homeostatic copper balance, resulting in copper deficiency ( Menkes disease) or copper overload ( Wilson disease), respectively. ATP7A and ATP7B exert their functions in copper transport through a variety of interdependent mechanisms and regulatory events, including their catalytic ATPase activity, copper-induced trafficking, post-translational modifications and protein - protein interactions. This paper reviews the extensive efforts that have been undertaken over the past few years to dissect and characterise these mechanisms, and how these are affected in Menkes and Wilson disease. As both disorders are characterised by an extensive clinical heterogeneity, we will discus how the underlying genetic defects correlate with the molecular functions of ATP7A and ATP7B and with the clinical expression of these disorders.

Strong interest in copper homeostasis is due to the fact that copper is simultaneously a catalytic co-factor of the vital enzymes, a participant in signaling, and a toxic agent provoking oxidative stress. In mammals, during development copper metabolism is conformed to two types. In embryonic type copper metabolism (ETCM), newborns accumulate copper to high level in the liver because its excretion via bile is blocked; and serum copper concentration is low because ceruloplasmin (the main copper-containing protein of plasma) gene expression is repressed. In the late weaning, the ETCM switches to the adult type copper metabolism (ATCM), which is manifested by the unlocking of copper excretion and the induction of ceruloplasmin gene activity. The considerable progress has been made in the understanding of the molecular basis of copper metabolic turnover in the ATCM, but many aspects of the copper homeostasis in the ETCM remain unclear. The aim of this study was to investigate the copper metabolism during transition from the ETCM (up to 12-days-old) to the ATCM in the rats. It was shown that in the liver, copper was accumulated in the nuclei during the first 5 days of life, and then it was re-located to the mitochondria. In parallel with the mitochondria, copper bulk bound with cytosolic metallothionein was increased. All compartments of the liver cells rapidly lost most of their copper on the 13th day of life. In newborns, serum copper concentration was low, and its major fraction was associated with holo-Cp, however, a small portion of copper was bound to extracellular metallothionein and a substance that was slowly eluted during gel-filtration. In adults, serum copper concentration increased by about a factor of 3, while metallothionein-bound copper level decreased by a factor of 2. During development, the expression level of Cp, Sod1, Cox4i1, Atp7b, Ctr1, Ctr2, Cox17, and Ccs genes was significantly increased, and metallothionein was decreased. Atp7a gene’s activity was fully repressed. The copper routes in newborns are discussed.

Menkes' disease is a rare X-linked multisystemic lethal disorder of copper transport metabolism. Failure of synthesis of several copper enzymes explains most of the clinical features, which were characterised by neurodegenerative symptoms and connective tissue manifestations. Most cases are still prone to rapidly progressive cerebral degeneration and early death in the first few years. Since CNS-dysfunction usually preceeds development of the pathognomonic "steely" hair, delay of clinical diagnosis and onset of therapeutic intervention precludes longlasting neurological benefit. This is particularly true for patients with large deletions or severe truncations of the responsible ATP7A gene. We report on our own experience with a patient, who was diagnosed to be affected by Menkes' syndrome at the age of one year, due to the specific hair texture and biochemical abnormalities. Molecular investigation revealed a total deletion of exon 15 of the ATP7A gene. Heterozygosity was confirmed by means of real-time PCR in the child's mother, but could be excluded in the grandmother and other female relatives at risk. Therapeutic support with subcutaneous injection of copper-histidinate normalised diminished copper and coeruloplasmin serum levels, but was unable to influence the clinical course and to prevent the fatal outcome at the age of two years. This observation is in line with the experience of the literature claiming that currently available medication will hardly be able to normalise brain copper levels. However, observations of clinical variants of Menkes' disease with quite a different outcome and, more importantly, emerging of alternative copper transport pathways might still justify this time-limited therapeutic intervention.

Expression of two copper-transporting P1-type ATPases (ATP7A and ATP7B), the CTR1 protein, a high-affinity copper transporter, and ceruloplasmin (Cp), a copper-containing ferroxidase was studied. The level of mRNA of these proteins was determined by RT-PCR analysis, the distribution of polypeptides encoded by these genes was determined by immunoblotting, and the type of cells expressing these genes was identified immunohistochemically. It was found that the major product of Cp gene in the brain is the cell membrane-bound Cp. Secretory Cp, whose molecule contains the greatest number of weakly associated copper atoms, is synthesized in the choroid plexus. CTR1 mRNA is evenly distributed in the brain; however, its content is twice higher in the vascular plexus. The Atp7a gene is active in all brain regions, whereas the Atp7b gene is active only in the hypothalamus. The membrane-bound Cp is expressed in glial cells of all types and in ependyma cells. ATP7B and ATP7A are expressed predominantly in ependymyocytes and neurons, respectively. The organization of copper transport in mammalian brain is discussed.

Menkes disease (MD) is an X-linked multisystemic lethal disorder of copper metabolism dominated by neurodegenerative symptoms and connective tissue disturbances. MD results from mutations in the ATP7A gene, which encodes a membrane-bound copper transporting P-type ATPase located in the trans-Golgi network. In this study we describe screening of 383 unrelated patients affected with Menkes disease for gross deletions in ATP7A gene and finding of 57 patients. The present data suggests that gross deletion of ATP7A is the disease-causing mutation in 14.9% of the Menkes disease patients. Except for a few cases, gross gene deletions result in the classical form of Menkes disease with death in early childhood.