Menkes disease is a lethal X-linked recessive neurodegenerative disorder of copper transport caused by mutations in ATP7A, which encodes a copper-transporting ATPase. Early postnatal treatment with copper injections often improves clinical outcomes in affected infants. While Menkes disease newborns appear normal neurologically, analyses of fetal tissues including placenta indicate abnormal copper distribution and suggest a prenatal onset of the metal transport defect. In an affected fetus whose parents found termination unacceptable and who understood the associated risks, we began in utero copper histidine treatment at 31.5 weeks gestational age. Copper histidine (900 μg per dose) was administered directly to the fetus by intramuscular injection (fetal quadriceps or gluteus) under ultrasound guidance. Percutaneous umbilical blood sampling enabled serial measurement of fetal copper and ceruloplasmin levels that were used to guide therapy over a four-week period. Fetal copper levels rose from 17 μg/dL prior to treatment to 45 μg/dL, and ceruloplasmin levels from 39 mg/L to 122 mg/L. After pulmonary maturity was confirmed biochemically, the baby was delivered at 35.5 weeks and daily copper histidine therapy (250 μg sc b.i.d.) was begun. Despite this very early intervention with copper, the infant showed hypotonia, developmental delay, and electroencephalographic abnormalities and died of respiratory failure at 5.5 months of age. The patient’s ATP7A mutation, which severely disrupted mRNA splicing, resulted in complete absence of ATP7A protein on Western blots. These investigations suggest that prenatally initiated copper replacement is inadequate to correct Menkes disease caused by severe loss-of-function mutations, and that postnatal ATP7A gene addition represents a rational approach in such circumstances.
Copper is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular copper metabolism, an elaborate system of chaperones and transporters has evolved, although no human copper chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited a homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the copper chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163 with tryptophan in domain II of CCS, which interacts directly with SOD1. Biochemical analyses of the patient’s fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal copper homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a copper chaperone.
CCS; SOD1; copper; chaperone
Fetal brain-directed gene addition represents an under-appreciated tool for investigating novel therapeutic approaches in animal models of central nervous system diseases with early prenatal onset. Choroid plexuses (CPs) are specialized neuroectoderm-derived structures that project into the brain's ventricles, produce cerebrospinal fluid (CSF), and regulate CSF biochemical composition. Targeting the CP may be advantageous for adeno-associated viral (AAV) gene therapy for central nervous system disorders due to its immunoprivileged location and slow rate of epithelial turnover. Yet the capacity of AAV vectors to transduce CP has not been delineated precisely. We performed intracerebroventricular injections of recombinant AAV serotype 5-green fluorescent protein (rAAV5-GFP) or rAAV9-GFP in embryonic day 15 (E15) embryos of CD-1 and C57BL/6 pregnant mice and quantified the percentages of GFP expression in CP epithelia (CPE) from lateral and fourth ventricles on E17, postnatal day 2 (P2), and P22. AAV5 was selective for CPE and showed significantly higher transduction efficiency in C57BL/6 mice (P = 0.0128). AAV9 transduced neurons and glial cells in both the mouse strains, in addition to CPE. We documented GFP expression in CPE on E17, within just 48 hours of rAAV administration to the fetal lateral ventricle, and expression by both the serotypes persisted at P130. Our results indicate that prenatal administration of rAAV5 and rAAV9 enables rapid, robust, and sustained transduction of mouse CPE and buttress the rationale for experimental therapeutics targeting the CP.
blood-cerebrospinal fluid barrier; choroid plexus; in utero gene therapy; rAAV5; rAAV9; survival surgery
The primary mechanism of copper transport to the brain is unknown, although this process is drastically impaired in Menkes disease, an X-linked neurodevelopmental disorder caused by mutations in an evolutionarily conserved copper transporter, ATP7A. Potential central nervous system entry routes for copper include brain capillary endothelial cells that originate from mesodermal angioblasts and form the blood-brain barrier, and the choroid plexuses, which derive from embryonic ectoderm, and form the blood-cerebrospinal fluid barrier. We exploited a rare (and first reported) example of somatic mosaicism for an ATP7A mutation to shed light on questions about copper transport into the developing brain. In a 20-month-old Menkes disease patient evaluated before copper treatment, blood copper and catecholamine concentrations were normal, whereas levels in cerebrospinal fluid were abnormal and consistent with his neurologically severe phenotype. We documented disparate levels of mosaicism for an ATP7A missense mutation, P1001L, in tissues derived from different embryonic origins; allele quantitation showed P1001L in approximately 27% and 88% of DNA samples from blood cells (mesoderm-derived) and cultured fibroblasts (ectoderm-derived), respectively. These findings imply that the P1001L mutation in the patient preceded formation of the three primary embryonic lineages at gastrulation, with the ectoderm layer ultimately harboring a higher percentage of mutation-bearing cells than mesoderm or endoderm. Since choroid plexus epithelia are derived from neuroectoderm, and brain capillary endothelial cells from mesodermal angioblasts, the clinical and biochemical findings in this infant support a critical role for the blood-CSF barrier (choroid plexus epithelia) in copper entry to the developing brain.
Somatic mosaicism; Menkes disease; ATP7A; copper metabolism; choroid plexus
ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7AT994I and ATP7AP1386S, with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7AT994I and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7AT994I mislocalization. Flow cytometry documented that non-permeabilized ATP7AP1386S fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.
ATP7A is a copper-transporting ATPase critical for central and peripheral nervous system function. Mutations in ATP7A cause Menkes disease and occipital horn syndrome (OHS), allelic X-linked recessive conditions that feature vascular abnormalities ascribed to low activity of lysyl oxidase, a copper-dependent enzyme. From a recently created Menkes disease/OHS patient registry, we identified 4 of 95 subjects with major congenital heart defects (4.2%), a proportion exceeding the general population prevalence (≈1%). In conjunction with mouse models of Menkes disease, OHS, and lysyl oxidase deficiency, which feature aortic aneurysms, irregular attachment between vascular endothelium and mesoderm and other defects of embryological development, our observation suggests an important role of copper metabolism in cardiac development. Congenital heart disease may be an under-appreciated abnormality in Menkes disease, and should be considered in a broad differential diagnosis of cardiac defects found prenatally in male fetuses. Conversely, newborn infants with suspected or confirmed Menkes disease should be evaluated for heart disease by careful clinical examination and echocardiography, if indicated.
ATP7A; congenital heart disease; lysyl oxidase; Menkes disease; occipital horn syndrome
Pediatric neck masses should trigger a high index of suspicion for certain genetic disorders of connective tissue. To highlight this, we report on three infants with Menkes disease, an inherited disorder of copper transport, who developed large, unilateral neck masses at between 7 and 17 months of age. All were identified in imaging studies as internal jugular phlebectasia. The masses, which enlarged on crying or exertion, have remained clinically benign in these patients for 20, 17 and 2 months, respectively. While arterial tortuosity and aneurysms have been reported often in Menkes disease, venous phlebectasia has rarely been described. We speculate that low activity of the copper-dependent enzyme, lysyl oxidase, leading to reduced tensile strength in the deep cervical fascia comprising the carotid sheath may predispose to internal jugular phlebectasia in these individuals. Improved survival and neurological outcomes in infants with Menkes disease due to advances in early diagnosis and treatment may be associated with recognition of novel clinical stigmata of this condition such as internal jugular phlebectasia.
internal jugular phlebectasia; venous aneurysm; neck mass; Menkes disease
Pronounced intrafamilial variability is unusual in Menkes disease and its variants. We report two unrelated families featuring affected members with unusually disparate clinical and biochemical phenotypes and explore the underlying molecular mechanisms.
We measured biochemical markers of impaired copper transport in five patients from two unrelated families and used RNase protection, quantitative reverse transcription (RT)‐PCR, Western blot analysis and yeast complementation studies to characterise two ATP7A missense mutations, A1362D and S637L.
In two brothers (family A) with A1362D, RNase protection and Western blot analyses revealed higher amounts of ATP7A transcript and protein in the older, mildly affected patient, who also had a higher plasma copper level and lower cerebrospinal fluid dihydroxyphenylalanine : dihydroxyphenylglycol ratio. These findings indicate greater gastrointestinal absorption of copper and higher activity of dopamine‐β‐hydroxylase, a copper‐dependent enzyme, respectively. In family B, three males with a missense mutation (S637L) in an exon 8 splicing enhancer showed equally reduced amounts of ATP7A transcript and protein by quantitative RT‐PCR and western blot analysis, respectively, despite a more severe phenotype in the youngest. This patient's medical history was notable for cardiac arrest as a neonate, to which we attribute his more severe neurodevelopmental outcome.
These families illustrate that genetic and non‐genetic mechanisms may underlie intrafamilial variability in Menkes disease and its variants.
Menkes disease; intrafamilial variation; gene expression, ATP7A; residual copper transport
Menkes disease is a fatal neurodegenerative disorder of infancy caused by defects in an X-linked copper transport gene, ATP7A. Evidence from a recent clinical trial indicates that favorable response to early treatment of this disorder with copper injections involves mutations that retain some copper transport capacity. In three unrelated infants, we identified the same mutation, G727R, in the second transmembrane segment of the ATP7A gene product that complemented a S. cerevisiae copper transport mutant, consistent with partial copper transport activity. Quantitative reverse transcription-polymerase chain reaction studies showed approximately normal levels of ATP7AG727R transcript in two patients’ fibroblasts compared to wild type controls, but Western blot analyses showed markedly reduced quantities of ATP7A protein, suggesting post-translational degradation. We confirmed the latter by comparing degradation rates of mutant and wild type ATP7A via cyclohexamide treatment of cultured fibroblasts; half-life of the G727R mutant was 2.9 hr and for the wild-type, 11.4 hr. We also documented a X-box binding protein 1 splice variant in G727R cells - known to be associated with the cellular misfolded protein response. Patient A, diagnosed 6 months of age, began treatment at 228 days (7.6 mos) of age. At his current age (2 years), his overall neurodevelopment remains at a 2 to 4 month level. In contrast, patients B and C were diagnosed in the neonatal period, began treatment within 25 days of age, and show near normal neurodevelopment at their current ages, 3 years (B), and 7 months (C). The poor clinical outcome in patient A with the same missense mutation as patients A and B with near normal oucomes, confirms the importance of early medical intervention in Menkes disease and highlights the critical potential benefit of newborn screening for this disorder.
Menkes disease is an X-linked recessive disorder of copper transport caused by mutations in ATP7A, a copper-transporting ATPase. Certain radiologic findings reported in this condition overlap with those caused by child abuse. However, cervical spine defects simulating cervical spine fracture, a known result of nonaccidental pediatric trauma, have not been reported previously in this illness.
To assess the frequency of cervical spine anomalies in Menkes disease after discovery of an apparent C2 posterior arch defect in a child participating in a clinical trial.
Materials and methods
We examined cervical spine radiographs obtained in 35 children with Menkes disease enrolled in a clinical trial at the National Institutes of Health Clinical Center.
Four of the 35 children with Menkes disease had apparent C2 posterior arch defects consistent with spondylolysis or incomplete/delayed ossification.
Defects in C2 were found in 11% of infants and young children with Menkes disease. Discovery of cervical spine defects expands the spectrum of radiologic findings associated with this condition. As with other skeletal abnormalities, this feature simulates nonaccidental trauma. In the context of Menkes disease, suspicions of child abuse should be considered cautiously and tempered by these findings to avoid unwarranted accusations.
Menkes disease; Cervical spine; Bone abnormalities; Copper metabolism
Menkes disease is a fatal neurodegenerative disorder of infancy caused by diverse mutations in a copper-transport gene, ATP7A. Early treatment with copper injections may prevent death and illness, but presymptomatic detection is hindered by the inadequate sensitivity and specificity of diagnostic tests. Exploiting the deficiency of a copper enzyme, dopamine-β-hydroxylase, we prospectively evaluated the diagnostic usefulness of plasma neurochemical levels, assessed the clinical effect of early detection, and investigated the molecular bases for treatment outcomes.
Between May 1997 and July 2005, we measured plasma dopamine, norepinephrine, dihydroxyphenylacetic acid, and dihydroxyphenylglycol in 81 infants at risk. In 12 newborns who met the eligibility criteria and began copper-replacement therapy within 22 days after birth, we tracked survival and neurodevelopment longitudinally for 1.5 to 8 years. We characterized ATP7A mutations using yeast complementation, reverse-transcriptase–polymerase-chain-reaction analysis, and immunohistochemical analysis.
Of 81 infants at risk, 46 had abnormal neurochemical findings indicating low dopamine-β-hydroxylase activity. On the basis of longitudinal follow-up, patients were classified as affected or unaffected by Menkes disease, and the neurochemical profiles were shown to have high sensitivity and specificity for detecting disease. Among 12 newborns with positive screening tests who were treated early with copper, survival at a median follow-up of 4.6 years was 92%, as compared with 13% at a median follow-up of 1.8 years for a historical control group of 15 late-diagnosis and late-treatment patients. Two of the 12 patients had normal neurodevelopment and brain myelination; 1 of these patients had a mutation that complemented a Saccharomyces cerevisiae copper-transport mutation, indicating partial ATPase activity, and the other had a mutation that allowed some correct ATP7A splicing.
Neonatal diagnosis of Menkes disease by plasma neurochemical measurements and early treatment with copper may improve clinical outcomes. Affected newborns who have mutations that do not completely abrogate ATP7A function may be especially responsive to early copper treatment.
Techniques for the diagnosis of copper transport disorders are increasingly important due to recent recognition of previously unappreciated clinical phenotypes and emerging advances in the treatment of these conditions. Here, we collate the diagnostic approaches and techniques currently employed for biochemical and molecular assessment of at-risk individuals in whom abnormal copper metabolism is suspected.
Epilepsy is a major feature of Menkes disease, an X-linked recessive infantile neurodegenerative disorder caused by mutations in ATP7A, which produces a copper-transporting ATPase. Three prior surveys indicated clinical seizures and electroencephalographic (EEG) abnormalities in a combined 27 of 29 (93%) symptomatic Menkes disease patients diagnosed at 2 months of age or older. To assess the influence of earlier, presymptomatic diagnosis and treatment on seizure semiology and brain electrical activity, we evaluated 71 EEGs in 24 Menkes disease patients who were diagnosed and treated with copper injections in early infancy (≤6 weeks of age), and whose ATP7A mutations we determined. Clinical seizures were observed in only 12.5% (3/24) of these patients, although 46% (11/24) had at least one abnormal EEG tracing, including 50% of patients with large deletions in ATP7A, 50% of those with small deletions, 60% of those with nonsense mutations, and 57% of those with canonical splice junction mutations. In contrast, five patients with mutations shown to retain partial function, either via some correct RNA splicing or residual copper transport capacity, had neither clinical seizures nor EEG abnormalities. Our findings suggest that early diagnosis and treatment improve brain electrical activity and decrease seizure occurrence in classical Menkes disease irrespective of the precise molecular defect. Subjects with ATP7A mutations that retain some function seem particularly well protected by early intervention against the possibility of epilepsy.
Protein translation ends when a stop codon in a gene’s messenger RNA transcript enters the ribosomal A site. Mutations that create premature stop codons (nonsense mutations) typically cause premature translation termination. An alternative outcome, read-through translation (or nonsense suppression), is well known in prokaryotic, viral, and yeast genes but has not been clearly documented in humans except in the context of pharmacological manipulations. Here, we identify and characterize native read-through of a nonsense mutation (R201X) in the human copper transport gene, ATP7A. Western blotting, in vitro expression analyses, immunohistochemistry, and yeast complementation assays using cultured fibroblasts from a classical Menkes disease patient all indicated small amounts of native ATP7AR201X read-through and were associated with a dramatic clinical response to early copper treatment.
Classical Menkes disease is an X-linked recessive neurodegenerative disorder caused by mutations in a P-type ATPase (ATP7A) that normally delivers copper to the developing central nervous system. Infants with large deletions, or other mutations in ATP7A that incapacitate copper transport to the brain, show poor clinical outcomes and subnormal brain copper despite early subcutaneous copper histidine (CuHis) injections. These findings suggest a need for direct central nervous system approaches in such patients. To begin to evaluate an aggressive but potentially useful new strategy for metabolic improvement of this disorder, we studied the acute and chronic effects of CuHis administered by intracerebroventricular (ICV) injection in healthy adult rats. Magnetic resonance imaging (MRI) after ICV CuHis showed diffuse T1-signal enhancement, indicating wide brain distribution of copper after ICV administration, and implying the utility of this paramagnetic metal as a MRI contrast agent. The maximum tolerated dose (MTD) of CuHis, defined as the highest dose that did not induce overt toxicity, growth retardation, or reduce lifespan, was 0.5 mcg. Animals receiving multiple infusions of this MTD showed increased brain copper concentrations, but no significant differences in activity, behavior, and somatic growth, or brain histology compared to saline-injected controls. Based on estimates of the brain copper deficit in Menkes disease patients, CuHis doses 10-fold lower than the MTD found in this study may restore proper brain copper concentration. Our results suggest that ICV CuHis administration have potential as a novel treatment approach in Menkes disease infants with severe mutations. Future trials of direct CNS copper administration in mouse models of Menkes disease will be informative.
Copper histidine; Intracerebral administration; Maximum tolerated dose; Menkes disease; Copper transport
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 (Mobr), a mouse model of Menkes disease. The cDNA sequence for ATOX1 is known, and the genomic organization has not been reported.
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.
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.
Menkes disease is an X-linked recessive neurodevelopmental disorder resulting from mutation in a copper-transporting ATPase gene. Menkes disease can be detected by relatively high concentrations of dopamine (DA) and its metabolites compared to norepinephrine (NE) and its metabolites, presumably because dopamine-beta-hydroxylase (DBH) requires copper as a co-factor. The relative diagnostic efficiencies of levels of catechol analytes, alone or in combination, in neonates at genetic risk of Menkes disease have been unknown.
Plasma from 44 at-risk neonates less than 30 days old were assayed for DA, NE, and other catechols. Of the 44, 19 were diagnosed subsequently with Menkes disease, and 25 were unaffected.
Compared to unaffected at-risk infants, those with Menkes disease had high plasma DA (P < 10−6) and low NE (P < 10−6) levels. Considered alone, neither DA nor NE levels had perfect sensitivity, whereas the ratio of DA:NE was higher in all affected than in all unaffected subjects (P = 2 × 10−8). Analogously, levels of the DA metabolite, dihydroxyphenylacetic acid (DOPAC), and the NE metabolite, dihydroxyphenylglycol (DHPG), were imperfectly sensitive, whereas the DOPAC:DHPG ratio was higher in all affected than in all unaffected subjects (P = 2 × 10−4). Plasma dihydroxyphenylalanine (DOPA) and the ratio of epinephrine (EPI):NE levels were higher in affected than in unaffected neonates (P = 0.0015; P = 0.013).
Plasma DA:NE and DOPAC:DHPG ratios are remarkably sensitive and specific for diagnosing Menkes disease in at-risk newborns. Affected newborns also have elevated DOPA and EPI:NE ratios, which decreased DBH activity alone cannot explain.
Menkes; Dopamine; Norepinephrine; Dopamine-β-hydroxylase; DHPG; DOPAC; Diagnosis