Clinical outcomes in Menkes disease patients with a copper-responsive ATP7A mutation, G727R

doi:10.1016/j.ymgme.2008.06.015

Molecular Genetics and Metabolism

Volume 95, Issue 3, November 2008, Pages 174-181

https://doi.org/10.1016/j.ymgme.2008.06.015 Get rights and content

Abstract

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 ATP7A that complemented a Saccharomyces cerevisiae copper transport mutant, consistent with partial copper transport activity. Quantitative reverse transcription-polymerase chain reaction studies showed approximately normal levels of ATP7A_G727R transcript in two patients’ fibroblasts compared to wild-type controls, but Western blot analyses showed markedly reduced quantities of ATP7A, 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 h and for the wild-type, 11.4 h. 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 months) of age. At his current age (2.5 years), his overall neurodevelopment remains at a 2- to 4-month level. In contrast, patient B and patient 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 (patient B), and 7 months (patient C). The poor clinical outcome in patient A with the same missense mutation as patient A and patient 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.

Section snippets

Patient A

This infant, of Korean heritage, and with no prior family history of Menkes disease, was born at term to a healthy 33-year-old G₂P₁A₀ mother. He was diagnosed as having Menkes disease at 6 months of age based on clinical phenotype, biochemical findings (low serum copper), and molecular testing by a commercial laboratory that revealed the G727R mutation. Despite his severe neurodevelopmental delays at the time of diagnosis, we enrolled him in a copper histidine treatment protocol as a special

Mutation analysis

In all three patients, a G to A transition at nucleotide 2324 in exon 10 of the ATP7A gene (Fig. 1) was present that altered the translational codon from GGA to AGA, predicting substitution of arginine (R) for glycine (G) at amino acid residue 727.

Yeast complementation assay

Yeast strains transformed with pYes6/CT vector carrying the human cDNA for wild-type and G727R ATP7A expressed the gene product, as assessed by Western blot analysis (Fig. 2). Yeast strains were plated on four different media; all strains grew on YPD,

Discussion

The G727R missense mutation in ATP7A may be relatively common defect, as it has now been reported in five unrelated patients with Menkes disease from the United States, the Far East [23], and Middle East. Most ATP7A alterations are private mutations unique to individual families; we speculate that the GC-rich sequence from bases 2323 to 2329 or poly-T sequence from bases 2318 to 2322 (Fig. 1) may predispose to the G to A transition at base 2324 during DNA replication. We also note that the

Acknowledgments

We are grateful to Sarah Godwin for help in mutation screening, Courtney Holmes and David Goldstein for catecholamine analysis, Christine Kaneski for cell culture, and Olga Protchenko and Caroline Philpott for advice on yeast Western blotting. This work was supported by the NICHD Intramural Research Program.

References (37)

H. Roelofsen et al.
Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion
Gastroenterology
(2000)
S.R. White et al.
Spectrum of EEG findings in Menkes disease
Electroencephalogr. Clin. Neurophysiol.
(1993)
S.G. Kaler et al.
Successful early copper therapy in Menkes disease associated with a mutant transcript containing a small in-frame deletion
Biochem. Mol. Med.
(1996)
J.R. Tang et al.
Functional copper transport explains neurologic sparing in occipital horn syndrome
Genet. Med.
(2006)
D.S. Grenda et al.
Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis
Blood
(2007)
D.J. Price et al.
Internal jugular phlebectasia in Menkes disease
Int. J. Pediatr. Otorhinolaryngol.
(2007)
D. Barker et al.
Restriction sites containing CpG show a higher frequency of polymorphism in human DNA
Cell
(1984)
A.S. Payne et al.
Functional expression of the Menkes disease protein reveals common biochemical mechanisms among the copper-transporting P-type ATPases
J. Biol. Chem.
(1998)
B.E. Kim et al.
A conditional mutation affecting localization of the Menkes disease copper ATPase. Suppression by copper supplementation
J. Biol. Chem.
(2002)
C. Vulpe et al.
Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase
Nat. Genet.
(1993)

J. Chelly J et al.

Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein

Nat. Genet.

(1993)

J.F.B. Mercer et al.

Isolation of a partial candidate gene for Menkes disease by positional cloning

Nat. Genet.

(1993)

S.G. Kaler et al.

Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at the MNK locus

Nat. Genet.

(1994)

S.G. Kaler SG et al.

Expression of the Menkes disease homolog in rodent neuroglial cells

Neurosci. Res. Commun.

(1998)

V.C. Cullota et al.

Disorders of copper transport

S.G. Kaler, Menkes disease, Adv. Pediatr. 41 (1994), L.A. Barnes (Ed.), C.V. Mosby, St. Louis, pp....

M.L. Schlief et al.

NMDA receptor activation mediates copper homeostasis in hippocampal neurons

J. Neurosci.

(2005)

M.L. Schlief et al.

Role of the Menkes copper-transporting ATPase in NMDA receptor-mediated neuronal toxicity

Proc. Natl. Acad. Sci. USA

(2006)

Cited by (36)

Copper Toxicity Associated With an ATP7A-Related Complex Phenotype
2021, Pediatric Neurology
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.
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.
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.
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 pathogenic mechanism in ATP7A-related dHMN.
Targeted next generation sequencing for newborn screening of Menkes disease
2020, Molecular Genetics and Metabolism Reports
Citation Excerpt :
The specific cost benefit of NBS for Menkes disease includes elimination of the extensive testing often involved in diagnostic odysseys of many affected infants (Table 1, Column 6). Evidentiary principles for clinical validity and clinical utility of genetic testing recently summarized by the Evaluation of Genomic Applications in Practice and Prevention (EGAPP) initiative [40] are fulfilled for ATP7A testing, based on multiple sources of evidence [17,19–23,29,31], as well as 2018 FDA designation of Fast Track status for Copper Histidinate treatment for patients diagnosed with classic Menkes disease who have not demonstrated significant clinical progression [41]. All infants found on NBS with ATP7A variants would be evaluated by plasma catecholamine analysis, an inexpensive and highly sensitive and reliable assay [17,31,42–44] to exclude a false positive diagnosis of Menkes disease.
Population-based newborn screening (NBS) allows early detection and treatment of inherited disorders. For certain medically-actionable conditions, however, NBS is limited by the absence of reliable biochemical signatures amenable to detection by current platforms. We sought to assess the analytic validity of an ATP7A targeted next generation DNA sequencing assay as a potential newborn screen for one such disorder, Menkes disease.
Dried blood spots from control or Menkes disease subjects (n = 22) were blindly analyzed for pathogenic variants in the copper transport gene, ATP7A. The analytical method was optimized to minimize cost and provide rapid turnaround time.
The algorithm correctly identified pathogenic ATP7A variants, including missense, nonsense, small insertions/deletions, and large copy number variants, in 21/22 (95.5%) of subjects, one of whom had inconclusive diagnostic sequencing previously. For one false negative that also had not been detected by commercial molecular laboratories, we identified a deep intronic variant that impaired ATP7A mRNA splicing.
Our results support proof-of-concept that primary DNA-based NBS would accurately detect Menkes disease, a disorder that fulfills Wilson and Jungner screening criteria and for which biochemical NBS is unavailable. Targeted next generation sequencing for NBS would enable improved Menkes disease clinical outcomes, establish a platform for early identification of other unscreened disorders, and complement current NBS by providing immediate data for molecular confirmation of numerous biochemically screened conditions.
Menkes disease and other ATP7A disorders
2020, Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease: Volume 1
Mutation of the copper transporter ATPase ATP7A, located in the trans-Golgi network and encoded by the X chromosome, causes the progressive copper deficiency disorder Menkes disease (MD) (also known as kinky hair disease), in addition to occipital horn syndrome and ATP7A-related distal motor neuropathy. ATP7A mediates intestinal Cu absorption to fulfill tissue requirements and prevents Cu over-accumulation. Regulation of ATP7A also contributes to Cu homeostasis in response to systemic Cu fluctuation. Basal levels of ATP7A are low in heart, spleen, and liver under Cu deficiency and administration of Cu restores normal ATP7A levels. In the intestine, ATP7A is regulated in the opposite manner, with decreases in systemic Cu increasing ATP7A and exogenous Cu increasing ATP7A, regulating Cu export into the circulation. The fundamental abnormality in the disease is thus the maldistribution of copper, which is unavailable as a cofactor of several enzymes including mitochondrial cytochrome c oxidase, lysyl oxidase, superoxide dismutase, dopamine beta-hydroxylase, and tyrosinase. Thus the main features of MD include mitochondrial respiratory chain dysfunction (complex IV deficiency), deficiency of collagen cross-links resulting in hair (pili torti and trichorrhexis nodosa) and vascular abnormalities (elongated cerebral vessels and subdural effusions), neuronal degeneration (markedly affecting Purkinje cells), and deficient melanin production dominate the disease manifestations. Affected neonates present with hypothermia, feeding difficulties, and seizures. The infants are pale and exhibit kinky hair. A variety of minimally symptomatic phenotypes, including ataxia or mental retardation have been recognized.
ATP7A Clinical Genetics Resource – A comprehensive clinically annotated database and resource for genetic variants in ATP7A gene
2020, Computational and Structural Biotechnology Journal
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.
Chelating principles in Menkes and Wilson diseases: Choosing the right compounds in the right combinations at the right time
2019, Journal of Inorganic Biochemistry
Citation Excerpt :
500 different ATP7A mutations have been observed and are unique in most families [20]. However, occurrence of the G727R mutation [21] in several unrelated Menkes patients [22] points to a mutation hot-spot at the gene level. Most mutations cause severe phenotypes, but some lead to a residual pump activity supporting a better although suboptimal loading of copper requiring enzymes [23,24].
Dysregulation of copper homeostasis in humans is primarily found in two genetic diseases of copper transport, Menkes and Wilson diseases, which show symptoms of copper deficiency or overload, respectively. However, both diseases are copper storage disorders despite completely opposite clinical pictures. Clinically, Menkes disease is characterized by copper deficiency secondary to poor loading of copper-requiring enzymes although sufficient body copper. Copper accumulates in non-hepatic tissues, but is deficient in blood, liver, and brain. In contrast, Wilson disease is characterized by symptoms of copper toxicity secondary to accumulation of copper in several organs most notably brain and liver, and a saturated blood copper pool. It is a challenge to correct copper dyshomeostasis in either disease though copper depletion in Menkes disease is most challenging. Both diseases are caused by defective copper export from distinct cells, and we seek to give new angles and guidelines to improve treatment of these two complementary diseases. Therapy of Menkes disease with copper-histidine, thiocarbamate, nitrilotriacetate or lipoic acid is discussed. In Wilson disease combination of a hydrophilic chelator e.g. trientine or dimercaptosuccinate with a brain shuttle e.g. thiomolybdate or lipoate, is discussed. New chelating principles for copper removal or delivery are outlined.
Cerebrospinal Fluid-Directed rAAV9-rsATP7A Plus Subcutaneous Copper Histidinate Advance Survival and Outcomes in a Menkes Disease Mouse Model
2018, Molecular Therapy Methods and Clinical Development
Citation Excerpt :
Moreover, sc copper histidinate injections are especially effective when commenced in the early, pre-symptomatic phase and when the ATP7A molecular defect does not completely abrogate copper transport.12 Certain Menkes disease patients whose ATP7A mutations were associated with residual copper transport not only averted premature death but attained normal neurodevelopmental outcomes in response to early sc copper histidinate treatment.12,17–21 However, affected patients harboring severe loss-of-function ATP7A mutations often respond less successfully to early intervention with sc copper histidinate alone.12,17,18,22,23
Menkes disease is a lethal neurodegenerative disorder of copper metabolism caused by mutations in an evolutionarily conserved copper transporter, ATP7A. Based on our prior clinical and animal studies, we seek to develop a therapeutic approach suitable for application in affected human subjects, using the mottled-brindled (mo-br) mouse model that closely mimics the Menkes disease biochemical and clinical phenotypes. Here, we evaluate the efficacy of low-, intermediate-, and high-dose recombinant adeno-associated virus serotype 9 (rAAV9)-ATP7A delivered to the cerebrospinal fluid (CSF), in combination with subcutaneous administration of clinical-grade copper histidinate (sc CuHis, IND #34,166). Mutant mice that received high-dose (1.6 × 10¹⁰ vg) cerebrospinal fluid-directed rAAV9-rsATP7A plus sc copper histidinate showed 53.3% long-term (≥300-day) survival compared to 0% without treatment or with either treatment alone. The high-dose rAAV9-rsATP7A plus sc copper histidinate-treated mutant mice showed increased brain copper levels, normalized brain neurochemical levels, improvement of brain mitochondrial abnormalities, and normal growth and neurobehavioral outcomes. This synergistic treatment effect represents the most successful rescue to date of the mo-br mouse model. Based on these findings, and the absence of a large animal model, we propose cerebrospinal fluid-directed rAAV9-rsATP7A gene therapy plus subcutaneous copper histidinate as a potential therapeutic approach to cure or ameliorate Menkes disease.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Published by Elsevier Inc.

Clinical outcomes in Menkes disease patients with a copper-responsive ATP7A mutation, G727R

Abstract

Section snippets

Patient A

Mutation analysis

Yeast complementation assay

Discussion

Acknowledgments

Gastroenterology

Electroencephalogr. Clin. Neurophysiol.

Biochem. Mol. Med.

Genet. Med.

Blood

Int. J. Pediatr. Otorhinolaryngol.

Cell

J. Biol. Chem.

J. Biol. Chem.

Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase

Nat. Genet.

Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein

Nat. Genet.

Isolation of a partial candidate gene for Menkes disease by positional cloning

Nat. Genet.

Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at the MNK locus

Nat. Genet.

Expression of the Menkes disease homolog in rodent neuroglial cells

Neurosci. Res. Commun.

Disorders of copper transport

NMDA receptor activation mediates copper homeostasis in hippocampal neurons

J. Neurosci.

Role of the Menkes copper-transporting ATPase in NMDA receptor-mediated neuronal toxicity

Proc. Natl. Acad. Sci. USA