Menkes Disease Patients Research Articles

Phenotypic and mutational spectrum of 17 Chinese patients with Menkes Disease.

Menkes Disease (MD) is a fatal X-linked recessive disorder caused by mutations in the ATP7A gene. Severe cases typically die before the age of three. Mild MD and occipital horn syndrome are variants of MD characterized by a less severe phenotype and longer survival. This case series aims to validate previous findings, expand the clinical phenotype, identify novel ATP7A mutations of MD patients. Observational data with follow-up were collected from 17 genetically diagnosed Chinese MD patients. All 17 patients exhibited neurological symptoms, including delayed motor milestones (100%) and seizures (58.8%). Unspecific pregnancy or delivery complications occurred in 9 patients (52.9%). The most prevalent connective tissue problems were abnormal hair (76.5%), followed by skeletal and dental abnormalities (52.9%), skin problems (41.2%) and hernia (35.3%). Sensorineural hearing loss (17.6%) was previously unreported. Coronary artery aneurysm and patent foramen ovale (5.9%) were infrequent. One 16-year-old boy carries pathological exon 3-4 deletion, presents novel mild phenotype including short stature and cerebellar ataxia. Out of 13 patients with follow-up (median: 24 months), 7 patients (53.8%) died with median survival of 40 months (range: 21-48 months), 3 patients (23.1%) show severe motor development delay and 2 (15.4%) have refractory epilepsy, only the mild MD patient shows improved cerebellar ataxia. Sixteen ATP7A mutations were identified including 6 small indels (37.5%), 5 nonsense mutations (31.2%), 2 missense mutations (12.5%), 2 exon deletions (12.5%), and 1 splice site mutation (6.25%). Fourteen mutations were novel. Our study further broadens the phenotypic and genotypic spectrums of Menkes disease.

EP195: Safety and efficacy of Copper Histidinate (CUTX-101) treatment for Menkes disease caused by severe loss-of-function variants in ATP7A

Menkes disease (MD) is a X-linked recessive disorder caused by variants in the copper transporter gene, ATP7A. Although MD patients appear asymptomatic at birth, they develop significant neurological signs and symptoms such as seizures, hypotonia, and failure to thrive approximately 6-10 weeks after birth. Death by 3 years of age is the typical outcome in untreated MD. Copper histidinate (CUTX-101) is being investigated for treatment of this illness.

ATP7A-Regulated Enzyme Metalation and Trafficking in the Menkes Disease Puzzle.

Copper is vital for numerous cellular functions affecting all tissues and organ systems in the body. The copper pump, ATP7A is critical for whole-body, cellular, and subcellular copper homeostasis, and dysfunction due to genetic defects results in Menkes disease. ATP7A dysfunction leads to copper deficiency in nervous tissue, liver, and blood but accumulation in other tissues. Site-specific cellular deficiencies of copper lead to loss of function of copper-dependent enzymes in all tissues, and the range of Menkes disease pathologies observed can now be explained in full by lack of specific copper enzymes. New pathways involving copper activated lysosomal and steroid sulfatases link patient symptoms usually related to other inborn errors of metabolism to Menkes disease. Additionally, new roles for lysyl oxidase in activation of molecules necessary for the innate immune system, and novel adapter molecules that play roles in ERGIC trafficking of brain receptors and other proteins, are emerging. We here summarize the current knowledge of the roles of copper enzyme function in Menkes disease, with a focus on ATP7A-mediated enzyme metalation in the secretory pathway. By establishing mechanistic relationships between copper-dependent cellular processes and Menkes disease symptoms in patients will not only increase understanding of copper biology but will also allow for the identification of an expanding range of copper-dependent enzymes and pathways. This will raise awareness of rare patient symptoms, and thus aid in early diagnosis of Menkes disease patients.

Neck masses due to internal jugular vein phlebectasia: Frequency in Menkes disease and literature review of 85 pediatric subjects.

Classic Menkes disease is a rare X-linked recessive disorder of copper metabolism caused by pathogenic variants in the copper transporter gene, ATP7A. Untreated affected individuals suffer failure to thrive and neurodevelopmental delays that begin at 6-8 weeks of age and progress inexorably to death, often within 3 years. Subcutaneous injections of Copper Histidinate (US Food and Drug Administration IND #34,166, Orphan product designation #12-3663) are associated with improved survival and neurological outcomes, especially when commenced within a month of birth. We previously identified internal jugular vein phlebectasia (IJP) in four Menkes disease subjects. This feature and other connective tissue abnormalities appear to be consequences of deficient activity of lysyl oxidase, a copper-dependent enzyme. Here, we report results from a prospective study of IJP based on 178 neck ultrasounds in 66 Menkes subjects obtained between November 2007 and March 2018. Nine patients met the criterion for IJP (one or more cross-sectional area measurements exceeding 2.2 cm2 ) and five subjects had clinically apparent neck masses that enlarged over time. Our prospective results suggest that IJP occurs in approximately 14% (9/66) of Menkes disease patients and appears to be clinically benign with no specific medical or surgical actionability. We surveyed the medical literature for prior reports of IJP in pediatric subjects and identified 85 individuals and reviewed the distribution of this abnormality by gender, sidedness, and underlying etiology. Taken together, Menkes disease accounts for 16% (15/94) of all reported IJP individuals. Neck masses from IJP represent underappreciated abnormalities in Menkes disease.

Diagnostic imaging features of Menkes disease

Objective To analyze the imaging features of Menkes disease in children, and in order to identify the diagnostic value and improve the knowledge of it. Methods The imaging findings of 9 Menkes disease patients confirmed by the clinical and laboratory examinations were retrospectively analyzed. All the patients were male aged from 2.0 to 7.0 months with a median age of 3.0 months. The clinical manifestations including seizures, psychomotor retardation, dystonia accompanied with light complexion and curly hair. The laboratory examinations showed low serum copper and ceruloplasmin levels. All the patients were performed with plain MR scan and MR angiography, 7 cases were performed with chest X-ray examinations and 4 cases were performed with bone X-ray examinations and 1 were performed with hand X-ray examinations. Results The plain MR scan showed white matter lesions in 8 cases, abnormality signal intensity of basal ganglia in 4 cases, subdural effusion in 2 cases. 8 cases had cerebral and cerebellar atrophy at different degrees. The MR angiography showed abnormal tortuosity of the main intracranial arteries. The chest X-ray of 5 in 7 cases showed that the front end of bilateral ribs were spherical and enlarged. 4 cases were performed with bone X-ray presenting, 3 out of 4 cases showed the widened metaphysis of tibia, fibula, femoral, humerus ulna and radius with the shape like cup-mouth and the formation of bony spur locally. The X ray examination of 1 case with connecting palm showed symphysodactylia. Conclusion Brain MR, X-ray of chest and bone showed imaging features of Menkes disease. MR angiography finding was the most distinctive feature, which has important value in the diagnosis of Menkes disease. Key words: Menkes disease; Magnetic resonance imaging; Copper

Identification of novel ATP7A mutations and prenatal diagnosis in Chinese patients with Menkes disease.

Menkes disease (MD) is a fatal X-linked multisystem disease caused by mutations in ATP7A. In this study, clinical and genetic analysis was performed in 24 male MD patients. Development delay, seizures, kinky coarse hair, and dystonia were found in 24, 22, 24, and 24 patients, respectively. Serum ceruloplasmin/copper tested in 19 patients was low. Abnormal classic features of MD presented in the MRI/MRA of 19 patients. Seventeen mutations of ATP7A were identified in 22 patients. Twelve were novel mutations including three small deletion/insertion, one missense mutation, two nonsense mutations, three splicing-site mutations, and three gross deletions. Twenty-two patients were genetically diagnosed; neither point mutation nor deletion/duplication was found in two of them. c.2179G>A found in five patients might be a hot-spot mutation. Prenatal molecular diagnosis was performed for five unrelated fetuses (1 female and 4 male), which found four fetuses to be wild type and one male carried the same mutation as the proband. This study of the largest sample of Chinese MD patients examined to date discovered the unique phenotype and genotype spectrum in Chinese patients with 12 novel mutations of ATP7A, and that c.2179G>A might be a hot-spot mutation in MD patients. Five successful prenatal diagnosis contributed important information for MD families.

Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

Menkes disease (MD) is caused by mutations in ATP7A, encoding a copper-transporting P-type ATPase which exhibits copper-dependent trafficking. ATP7A is found in the Trans-Golgi Network (TGN) at low copper concentrations, and in the post-Golgi compartments and the plasma membrane at higher concentrations. Here we have analyzed the effect of 36 ATP7A missense mutations identified in phenotypically different MD patients. Nine mutations identified in patients with severe MD, virtually eliminated ATP7A synthesis, in most cases due to aberrant RNA splicing. A group of 21 predominantly severe mutations led to trapping of the protein in TGN and displayed essentially no activity in a yeast-based functional assay. These were predicted to inhibit the catalytic phosphorylation of the protein. Four mutants showed diffuse post-TGN localization, while two displayed copper dependent trafficking. These six variants were identified in patients with mild MD and typically displayed activity in the yeast assay. The four post-TGN located mutants were presumably affected in the catalytic dephosphorylation of the protein. Together these results indicate that the severity of MD correlate with cellular localization of ATP7A and support previous studies indicating that phosphorylation is crucial for the exit of ATP7A from TGN, while dephosphorylation is crucial for recycling back to TGN.

The Involvement of Copper Transport in Von Willebrand Factor Multimer Assembly

In the event of vascular injury, the underlying prothrombotic extracellular matrix becomes exposed to circulating blood. Endothelial cells at the site of injury secrete multimeric von Willebrand factor (VWF) that will bind to the exposed extracellular matrix and create a scaffold to which platelets can adhere, forming a platelet plug. These critical steps after vascular injury are essential for creating a stable clot. Intracellular VWF multimer assembly is required for its extracellular function, and defects in VWF assembly cause the bleeding diathesis, von Willebrand disease. In a cell-free in vitro system, we and others find that EDTA or EGTA inhibits multimer formation, and calcium ions have been proposed to be required for this process. However, we find that the binding affinity of calcium ions for VWF D domains is very weak (Kdof 300-400 μM) and that buffers containing less than 2 μM calcium ions support multimer assembly even though few calcium binding sites could be occupied. These results suggest a role for cations other than calcium in VWF multimer assembly. Here we show that copper ions support VWF multimer assembly in a cell-free system and that the intracellular copper transporter, ATP7A, is required for VWF multimer assembly in cultured cells.Some calcium binding proteins undergo a conformational change upon binding calcium ions and exhibit a decrease in the apparent molecular weight (an electrophoretic mobility shift) as assessed by SDS-PAGE and Western blot. Although the D domains of VWF bind calcium ions, they do not exhibit this behavior. We tested a panel of other divalent cations including: iron, magnesium, manganese, zinc, and copper. We found that the VWF N-terminal D’D3 dimer bound copper ions as demonstrated by a shift in electrophoretic mobility. We therefore assessed the effect of copper ions on VWF multimer assembly in vitro. Briefly, addition of monensin (1.5 μM) to human umbilical vein endothelial cells (HUVEC) caused secretion of C-terminal dimers of proVWF into the media. Pretreatment of the conditioned media with the divalent cation chelator, EGTA, or the copper chelator, tetraethylenepentamine (TEPA), preceded immunopurification of C-terminal dimers of proVWF using a rabbit polyclonal anti-hVWF antibody (DAKO A0082) bound to protein A magnetic beads. Beads were incubated at pH 7.4 or pH 5.8, and in the presence or absence of divalent cations for 3 hours at 37°C before analyzing the VWF by gel electrophoresis. Addition of calcium ions (100 μM) to proVWF dimers pretreated with only EGTA allowed some VWF multimer assembly; however, copper ions (100 μM) induced the assembly of higher molecular weight multimers from proVWF dimers pretreated with either EGTA or TEPA. With either divalent cation, multimers assembled at low pH, but not at neutral pH. These findings support a role for copper ions in VWF synthesis.In vivo, free copper ions are toxic; therefore, they are sequestered by a system of chaperones and transporters. We examined the role of the intracellular copper transporter, ATP7A, in VWF multimerization because it is expressed in the trans Golgi network of endothelial cells, the site of VWF multimer assembly. Mick Petris (University of Missouri-Columbia) provided both wildtype and ATP7A knockout mouse embryonic fibroblasts (MEFs). We stably expressed full length human VWF and analyzed the secreted multimers by agarose gel electrophoresis. Wildtype MEFs produced multimeric VWF; however, culturing the MEFs in the presence of the copper chelator, TEPA, markedly impaired VWF multimer assembly. Moreover, ATP7A-null MEFs secreted only VWF dimers. These results indicate that copper ions and ATP7A contribute to VWF multimer assembly in cells.The dependence of VWF assembly on ATP7A in cultured cells suggests that humans with congenital ATP7A deficiency (Menkes disease) could have detectable abnormalities in VWF multimer structure, although dietary copper and parenteral copper replacement therapy could affect this phenotype. Our preliminary analyses have identified a Menkes disease patient with low plasma levels of VWF antigen, multimers and activity. Further study will be required to determine the prevalence and significance of altered VWF multimer assembly in Menkes disease. DisclosuresSadler:BioMarin: Consultancy; 23andMe: Consultancy; Ablynx: Consultancy.

Diagnostic copper imaging of Menkes disease by synchrotron radiation-generated X-ray fluorescence analysis.

Copper (Cu) is an indispensable metal for normal development and function of humans, especially in central nervous system (CNS). However, its redox activity requires accurate Cu transport system. ATP7A, a main Cu2+ transporting-ATPase, is necessary to efflux Cu across the plasma membrane and synthesize cuproenzymes. Menkes disease (MD) is caused by mutations in ATP7A gene. Clinically, MD is Cu deficiency syndrome and is treated with Cu-histidine injections soon after definite diagnosis. But outcome of the most remains poor. To estimate the standard therapy, Cu distribution in the treated classic MD patients is analyzed by synchrotron-generated X-ray fluorescence technique (SR-XRF), which identifies and quantifies an individual atom up to at subcellular level of resolution with wide detection area. SR-XRF analysis newly reveals that Cu exists in spinal cord parenchyma and flows out via venous and lymph systems. By systemic analysis, excess Cu is detected in the proximal tubular cells of the kidney, the mucosal epithelial cells of the intestine, and the lymph and venous systems. The current study suggests that the standard therapy supply almost enough Cu for patient tissues. But given Cu passes through the tissues to venous and lymph systems, or accumulate in the cells responsible for Cu absorption.

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

Is It a Pathogenic ATP7A Variation and Is It Menkes Disease?

This paper is a comment on “Menkes Disease Presenting with Epilepsia Partialis Continua.” While updating the LOVD (Leiden Open Variation Database) ATP7A mutation database (https://grenada.lumc.nl/LOVD2/MD/home.php) I came across a case described by Rizk and colleagues and published in your journal [1]. Rizk and colleagues report a 17-month-old girl who was referred to their hospital with continuous right hand movements which developed suddenly at age of 14 months, regression of milestones, and repetitive episodes of hypothermia. She had hypotonia and constipation. Brain magnetic resonance imaging showed among others cortical and cerebral atrophy. EEG was abnormal and she was diagnosed with epilepsia partialis continua. Magnetic resonance angiography showed relatively tortuous but patent intracranial vessels, with appearance of “hair pin” sign. She had normal hair also with light microscopy. Blood copper and ceruloplasmin levels were normal. The patient was born full term and had normal birth measures. Genetic testing revealed presence of a heterozygous variation, c.1138G>A [p.(Val380Met)], and the authors concluded the diagnosis of Menkes disease (MD) in this patient. Even though the clinical features of this female patient overlap with some of the features observed in Menkes disease patients, she lacks some of the major pathogenic criteria (such as reduced serum copper and ceruloplasmin levels, abnormal hair, hypopigmentation, and cutis laxa) (review in [2]) questioning the clinical diagnosis of Menkes disease, even though female patients may be less severely affected. Furthermore, the ATP7A variation identified is not convincing enough to establish a genetic diagnosis. Firstly, this variation is described at least once in a healthy individual (rs149523862, dbSNP database of short genetic variations, http://www.ncbi.nlm.nih.gov/SNP/), which questions its pathogenicity especially when taking into consideration that MD is a Mendelian disorder. This variation is a missense substitution in exon 4 of ATP7A proximal to but not within the 4th MBD (metal binding domain) of the ATP7A protein. Until now 70 different missense mutations in ATP7A have been described in 94 unrelated patients and none of these mutations were within the first 7 exons of ATP7A encoding the six MBDs [3]. It is generally believed that variations within these regions are more acceptable and do not necessarily lead to MD [3]. Furthermore, two commonly used and publicly available pathogenicity prediction methods, PhD-SNP (http://snps.biofold.org/phd-snp/phd-snp.html) and nsSNP analyser (http://snpanalyzer.uthsc.edu/), categorize this variation as neutral. Secondly, MD is an X-linked recessive disorder and manifesting heterozygotes are rare [4]. It is therefore extremely important to investigate the inheritance of this mutation and to perform X-inactivation studies before evaluating the clinical effect of this variation. In conclusion, the clinical diagnosis of this patient and the pathogenicity of the identified ATP7A variation should be reevaluated.

Changes in body weight and height in survivors of Menkes disease

ObjectiveTo explore the changes in the body weight and height of Menkes disease (MNK) patients treated with long-term copper–histidine. MethodsA survey involving a retrospective review of medical records or summaries of MNK patients was conducted. Patients were 44 males born after 1994, and their feeding method and genetic analysis of the ATP7A gene were reviewed. We compared the data of body weight and height from birth until 6 years between classical MNK patients and the general population obtained from national data and between patients who received early treatment and patients who received late treatment. ResultsAlthough five patients who received early treatment reached some developmental milestones, the body weight and height did not differ from patients who received late treatment in the mode of oral nutrition, and were lower in comparison to the national data (<3 percentile). ConclusionWe reported changes in the body weight and height of MNK patients who received early and late treatment. Although early treatment with copper–histidine had favorable effects on neurological development, it did not result in improvements in body weight and height. We suggest that the establishment of sufficient nutritional support is necessary along with early parenteral copper treatment to improve whole body condition in MNK patients.

Novel mutations and clinical outcomes of copper-histidine therapy in Menkes disease patients.

Menkes disease is a very rare X-linked copper metabolism disorder that results from an ATP7A gene mutation. With the advent of subcutaneous copper-histidine therapy, the early diagnosis of Menkes disease becomes of utmost importance for patients' prognosis. In the present study, the clinical characteristics of 12 Korean patients with Menkes disease (11 males and 1 female from 11 unrelated families) were described along with the mutation spectrum. Only 2 male patients were diagnosed in the neonatal period, and the other male patients were diagnosed at age 4.3 ± 1.9 months. The presenting signs included depigmented kinky hair, neurologic deficits, and hypotonia. Serum copper and ceruloplasmin levels were markedly decreased. Intracranial vessels were dilated with tortuosity and accompanied by regional cerebral infarctions, even at an early age. Of note, the female patient was diagnosed at age 18 months, during the evaluation for developmental delay, by characteristic MRA findings, biochemical profiles, and genetic evaluation. A total of 11 ATP7A mutations were identified, including five previously unreported mutations. Most mutations were truncated (except 1 missense mutation), including 3 frameshift, 2 nonsense, 3 large deletion, and 2 splice-site variants. The age at commencement of copper-histidine treatment was variable among patients age 7.3 ± 7.5 (0.5-27) months. Despite the treatment, seven patients died before age 5 years, and the remaining patients were severely retarded in neurodevelopment. The poor outcomes of our patients might be related to delayed therapy, but severe ATP7A mutations should be noted as well.

Menkes disease in Korea: ATP7A mutation and epilepsy phenotype.

Menkes disease (MD) is an X-linked recessive disorder characterized by progressive neuro-degeneration. There are few reports of epilepsy and electroencephalography (EEG) findings and few reports of MD patients in Korea. We explored MD genotypes and phenotypes, including epilepsy, in Korean patients. All patients diagnosed as MD in our hospital, seven males, were included in this study. Their medical records and EEG findings were reviewed retrospectively. All male patients had developmental delay/regression with hypotonia, and the appearance of their hair and skin was characteristic of MD. A recurrent missense mutation was found in two patients. Two nonsense mutations and one gross deletion were also found. The five male patients with identified molecular defects experienced anticonvulsant-resistant seizures. EEGs in focal seizures usually revealed interictal focal epileptiform discharges over the posterior region without focal slowing. This was followed by modified hypsarrhythmia with less polymorphic background activity in spasms and anteriorly dominant diffuse slowing with generalized and multifocal epileptiform discharges in myoclonic or generalized tonic seizures. Two patients with the same G727R missense mutation both developed seizures that evolved with age but differed in severity. G727R missense mutation may be relatively common in Korea, as in other countries. There was no clear correlation of genotype with phenotype, even in epilepsy and EEG abnormalities.

Modeling of Menkes disease via human induced pluripotent stem cells

Menkes disease (MD) is a copper-deficient neurodegenerative disorder that manifests severe neurologic symptoms such as seizures, lethargic states, and hypotonia. Menkes disease is due to a dysfunction of ATP7A, but the pathophysiology of neurologic manifestation is poorly understood during embryonic development. To understand the pathophysiology of neurologic symptoms, molecular and cellular phenotypes were investigated in Menkes disease-derived induced pluripotent stem cells (MD-iPSCs). MD-iPSCs were generated from fibroblasts of a Menkes disease patient. Abnormal reticular distribution of ATP7A was observed in MD-fibroblasts and MD-iPSCs, respectively. MD-iPSCs showed abnormal morphology in appearance during embryoid body (EB) formation as compared with wild type (WT)-iPSCs. Intriguingly, aberrant switch of E-cadherin (E-cad) to N-cadherin (N-cad) and impaired neural rosette formation were shown in MD-iPSCs during early differentiation. When extracellular copper was chelated in WT-iPSCs by treatment with bathocuprione sulfate, aberrant switch of E-cad to N-cad and impaired neuronal differentiation were observed, like in MD-iPSCs. Our results suggest that neurological defects in Menkes disease patients may be responsible for aberrant cadherin transition and impaired neuronal differentiation during early developmental stage.

Live-cell imaging of compartment-specific redox changes in menkes disease fibroblasts

Background Copper is an essential micronutrient and its misbalance in the body is associated with severe neurodegenerative disorders. While the importance of copper in the body is evident, mechanisms by which copper misbalance induces pathologic changes and disease symptoms are poorly understood. In this study, using fibroblasts from Menkes disease patient, as a cellular model of copper accumulation, we examined whether excess copper triggers specific and distinct changes in the redox environment of different cellular compartments. Subjects and Methods Skin fibroblasts from Menkes disease patient (YS cells, ATP7A -/- ) and his heterozygous mother (ATP7A +/- ) were used as an experimental system. Glutathione mediated redox environment and levels of H2O2 were investigated in nuclei, cytosol, and mitochondria of live cells by tagging the respective ratiometric sensors (GRX-roGFP and HyPer) with a compartment-specific localization signal. Results Under basal conditions, the YS and XS cells show similar glutathione mediated redox environment in the nucleus and cytosol. However, the mitochondria are oxidizing in YS cells. YS cells were observed to accumulate higher level of peroxide in the cytosol and mitochondria. We also found that copper accumulation in cytosol and nuclei of YS cells sensitize cells to glutathione depletion suggesting importance of glutathione in protection of cells against copper overload Conclusion Our experiments revealed differential response of cellular compartments to excess copper in cells. Nuclei, in spite of being a site of copper accumulation, do not show marked redox changes, suggesting presence of robust protective mechanisms operating in this compartment. H2O2 accumulation in cytosol in YS cells does not change glutathione balance, whereas mitochondria appear most affected, since both H2O2 levels and glutathione balance are altered. We propose that mitochondria could be a primary site of copper toxicity in Menkes fibroblasts. Molecular mechanisms underlying differential redox responses are presently being investigated.

Occipital horn syndrome and classical Menkes Syndrome caused by deep intronic mutations, leading to the activation of ATP7A pseudo-exon

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.

An Overview and Update ofATP7AMutations Leading to Menkes Disease and Occipital Horn Syndrome

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.

What can flies tell us about copper homeostasis?

Copper (Cu) is an essential redox active metal that is potentially toxic in excess. Multicellular organisms acquire Cu from the diet and must regulate uptake, storage, distribution and export of Cu at both the cellular and organismal levels. Systemic Cu deficiency can be fatal, as seen in Menkes disease patients. Conversely Cu toxicity occurs in patients with Wilson disease. Cu dyshomeostasis has also been implicated in neurodegenerative disorders such as Alzheimer's disease. Over the last decade, the fly Drosophila melanogaster has become an important model organism for the elucidation of eukaryotic Cu regulatory mechanisms. Gene discovery approaches with Drosophila have identified novel genes with conserved protein functions relevant to Cu homeostasis in humans. This review focuses on our current understanding of Cu uptake, distribution and export in Drosophila and the implications for mammals.

Effect of copper and disulfiram combination therapy on the macular mouse, a model of Menkes disease

Menkes disease (MD) is a genetic neurodegenerative disorder characterized by copper deficiency due to a defect in ATP7A. Standard treatment involves parenteral copper–histidine administration. However, the treatment is ineffective if initiated after two months of age, because the administered copper accumulates in the blood–brain barrier and is not transported to neurons. To resolve this issue, we investigated the effects of a combination therapy comprising copper and disulfiram, a lipophilic chelator, in the macular mouse, an animal model of MD. Seven-day-old macular mice treated subcutaneously with 50μg of CuCl2 on postnatal day 4 were used. The mice were given a subcutaneous injection of CuCl2 (10μg) with oral administration of disulfiram (0.3mg/g body weight) twice a week until eight weeks of age, and then sacrificed. Copper concentrations in the cerebellum, liver, and serum of treated macular mice were significantly higher than those of control macular mice, which received only copper. Mice treated with the combination therapy exhibited higher cytochrome c oxidase activity in the brain. The ratios of noradrenaline and adrenaline to dopamine in the brain were also increased by the treatment, suggesting that dopamine β-hydroxylase activity was improved by the combination therapy. Liver and renal functions were almost normal, although renal copper concentration was higher in treated macular mice than in controls. These results suggest that disulfiram facilitates the passage of copper across the blood–brain barrier and that copper–disulfiram combination therapy may be an effective treatment for MD patients.