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Stephen G Kaler - One of the best experts on this subject based on the ideXlab platform.
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targeted next generation sequencing for newborn screening of Menkes Disease
Molecular genetics and metabolism reports, 2020Co-Authors: Richard B Parad, Stephen G Kaler, Evan Mauceli, Tanya Sokolsky, Arindam BhattacharjeeAbstract:Abstract Purpose 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. Methods 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. Results 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. Conclusions 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.
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estimated birth prevalence of Menkes Disease and atp7a related disorders based on the genome aggregation database gnomad
Molecular genetics and metabolism reports, 2020Co-Authors: Stephen G Kaler, Carlos Ferreira, Lung S YamAbstract:Abstract Background Previous estimates of the prevalence of Menkes Disease, a lethal X-linked recessive disorder of copper metabolism, were based on confirmed clinical cases ascertained from specific populations and varied from 1 in 40,000 to 1 in 354,507. With newly available population-based allelic frequencies of DNA sequence variants, the expected birth prevalence of Menkes Disease and other ATP7A-related phenotypes can be reconsidered using Hardy-Weinberg theoretical principles. Methods We reviewed the canonical ATP7A transcript in the current version of gnomAD (v2.1.1) to evaluate frequency of complete loss-of-function alleles in a diverse normal control population. As a comparator, we used the DMD locus, associated with Duchenne and Becker Muscular Dystrophy, another X-linked recessive trait. We applied Hardy-Weinberg theory and PolyPhen-2 in silico plus REVEL and CADD ensemble analyses to calculate estimated frequencies of normal and predicted deleterious ATP7A alleles. Results We identified 1106 total ATP7A variants out of 205,523 alleles in gnomAD, with missense variants most common (43.4%). Complete loss-of-function variants were found in four ATP7A alleles (frequency = 0.0000194), including three frameshift/nonsense mutations and one canonical splice donor site defect. Assuming Hardy-Weinberg equilibrium, this frequency of pathogenic alleles predicts 1 in 34,810 live male births with Menkes Disease or other ATP7A-related disorders each year in the US. The same analysis for DMD loss-of-function variants predicted 1 in 7246 newborn males with Duchenne (or Becker) muscular dystrophy. We also identified nine ATP7A missense variants in gnomAD predicted as deleterious by PolyPhen-2 and stringent REVEL/CADD criteria, comprising 12 more Disease-causing alleles and raising the estimated birth prevalence to 1 in 8664 and predicting 225 newborns with Menkes Disease or other ATP7A-related disorders per year in the US alone. Conclusions Assuming Hardy-Weinberg equilibrium, the allelic frequency of deleterious ATP7A variants in a genomic database from a large diverse population predicts a birth prevalence of Menkes Disease or ATP7A-related disorders as high as 1 in 8664 live male births. This genome-driven ascertainment of deleterious ATP7A alleles in the population implies a higher birth prevalence of Menkes Disease and ATP7A-related conditions than previously appreciated. A population-based newborn screening pilot study for Menkes Disease will be instrumental in confirming the prediction.
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neck masses due to internal jugular vein phlebectasia frequency in Menkes Disease and literature review of 85 pediatric subjects
American Journal of Medical Genetics Part A, 2020Co-Authors: Stephen G Kaler, Kristen E Stevens, Julienne E Price, Jamie MarkoAbstract: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.
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Menkes Disease and other disorders related to atp7a
2019Co-Authors: Cynthia Abou Zeid, Stephen G KalerAbstract:Abstract The ATP7A-related disorders are a heterogeneous group of Diseases resulting from ATP7A (Menkes ATPase) dysfunction. They have varying phenotypes and clinical Diseases, depending on the underlying genetic mutations. Some ensue from direct changes in the gene that encodes the ATP7A protein. The severity of the mutations can affect the phenotype expressed. If the mutations are severe loss-of-function mutations, the result is a lethal neurodevelopmental and systemic Disease with low copper and reduced cuproenzymes activity (Menkes Disease). If the mutations are milder, such as with leaky splice junction defects, some copper transport is possible and the phenotype is less severe, as in occipital horn syndrome. Two unique missense mutations that do not impair the copper transport function of ATP7A but rather its trafficking, cause a motor neuron-specific illness that develops gradually, with no evidence of abnormal copper metabolism. ATP7A function can also be affected by mutations outside of its coding gene, such as in MEDNIK and Huppke–Brendel syndromes. The resulting defects in intracellular localization of the ATP7A (and ATP7B) pump(s) can impair normal copper transport function.
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cerebrospinal fluid directed raav9 rsatp7a plus subcutaneous copper histidinate advance survival and outcomes in a Menkes Disease mouse model
Molecular therapy. Methods & clinical development, 2018Co-Authors: Marie Reine Haddad, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Jose A Centeno, Eun-young Choi, Patricia M. Zerfas, Diego Martinelli, Martina Ralle, Stephen G KalerAbstract: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.
David S Goldstein - One of the best experts on this subject based on the ideXlab platform.
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cerebrospinal fluid directed raav9 rsatp7a plus subcutaneous copper histidinate advance survival and outcomes in a Menkes Disease mouse model
Molecular therapy. Methods & clinical development, 2018Co-Authors: Marie Reine Haddad, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Jose A Centeno, Eun-young Choi, Patricia M. Zerfas, Diego Martinelli, Martina Ralle, Stephen G KalerAbstract: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.
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Cerebrospinal Fluid-Directed rAAV9-rsATP7A Plus Subcutaneous Copper Histidinate Advance Survival and Outcomes in a Menkes Disease Mouse Model
Elsevier, 2018Co-Authors: Marie Reine Haddad, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Jose A Centeno, Eun-young Choi, Patricia M. Zerfas, Diego Martinelli, Martina RalleAbstract: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. Keywords: adeno-associated virus, Menkes Disease, ATP7A, copper, dopamine-beta-hydroxylase, choroid plexus epitheli
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tandem duplication of exons 1 7 neither impairs atp7a expression nor causes a Menkes Disease phenotype
JIMD reports, 2015Co-Authors: Eun-young Choi, Courtney Holmes, David S Goldstein, Marie Reine Haddad, Keyur Patel, Amalia Dutra, Evgenia Pak, Stephen G KalerAbstract:ATP7A duplications are estimated to represent the molecular cause of Menkes Disease in 4–10% of affected patients. We identified a novel duplication of ATP7A exons 1–7 discovered in the context of a challenging prenatal diagnostic situation. All other reported ATP7A duplications (n = 24) involved intragenic tandem duplications, predicted to disrupt the normal translational reading frame and produce nonfunctional ATP7A proteins. In contrast, the exon 1–7 duplication occurred at the 5′ end of the ATP7A gene rather than within the gene and did not correspond to any known copy number variants. We hypothesized that, if the exon 1–7 duplication was in tandem, functional ATP7A molecules could be generated depending on promoter selection, mRNA splicing, and the proximal and distal duplication breakpoints and that Menkes Disease would be averted. Here, we present detailed molecular characterization of this novel duplication, as well as 2-year postnatal clinical and biochemical correlations. The case highlights the ongoing need for cautious interpretation of prenatal genetic test results.
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l threo dihydroxyphenylserine corrects neurochemical abnormalities in a Menkes Disease mouse model
Annals of Neurology, 2013Co-Authors: Anthony Donsante, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Stephen G KalerAbstract:Objective Menkes Disease is a lethal neurodegenerative disorder of infancy caused by mutations in a copper-transporting ATPase gene, ATP7A. Among its multiple cellular tasks, ATP7A transfers copper to dopamine-beta-hydroxylase (DBH) within the lumen of the Golgi network or secretory granules, catalyzing the conversion of dopamine to norepinephrine. In a well-established mouse model of Menkes Disease, mottled-brindled, we tested whether systemic administration of L-threo-dihydroxyphenylserine (L-DOPS), a drug used successfully to treat autosomal recessive norepinephrine deficiency, would improve brain neurochemical abnormalities and neuropathology.
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in utero copper treatment for Menkes Disease associated with a severe atp7a mutation
Molecular Genetics and Metabolism, 2012Co-Authors: Marie Reine Haddad, Courtney Holmes, David S Goldstein, Charles J Macri, Beryl E Jacobson, Jose A Centeno, Edwina J Popek, W Gahl, Stephen G KalerAbstract: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 (Q724H), 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.
Marie Reine Haddad - One of the best experts on this subject based on the ideXlab platform.
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cerebrospinal fluid directed raav9 rsatp7a plus subcutaneous copper histidinate advance survival and outcomes in a Menkes Disease mouse model
Molecular therapy. Methods & clinical development, 2018Co-Authors: Marie Reine Haddad, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Jose A Centeno, Eun-young Choi, Patricia M. Zerfas, Diego Martinelli, Martina Ralle, Stephen G KalerAbstract: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.
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Cerebrospinal Fluid-Directed rAAV9-rsATP7A Plus Subcutaneous Copper Histidinate Advance Survival and Outcomes in a Menkes Disease Mouse Model
Elsevier, 2018Co-Authors: Marie Reine Haddad, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Jose A Centeno, Eun-young Choi, Patricia M. Zerfas, Diego Martinelli, Martina RalleAbstract: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. Keywords: adeno-associated virus, Menkes Disease, ATP7A, copper, dopamine-beta-hydroxylase, choroid plexus epitheli
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356 high resolution x ray fluorescence microscopy xfm indicates enhanced brain copper delivery in aav9 treated Menkes Disease mice
Molecular Therapy, 2016Co-Authors: Marie Reine Haddad, Patricia M. Zerfas, Martina Ralle, David Vine, Stephen G KalerAbstract:Menkes Disease is a lethal infantile neurodegenerative disorder caused by mutations in a copper transporter, ATP7A. The mottled-brindled (mo-br) mouse recapitulates the symptoms and early demise (14 days) of untreated human Menkes Disease (3 years). We previously demonstrated that viral gene therapy using cerebrospinal fluid (CSF)-directed AAV9-ATP7A in combination with subcutaneous (sc) clinical grade copper histidinate enhanced survival, normalized growth, and improved biochemical and neurobehavioral outcomes in this model. In the current study, we examined copper biodistribution and concentration in specific brain regions for treated and untreated mutants compared to wild type mice, using X-ray fluorescence microscopy (XFM). XFM enables sensitive, quantitative measurement of the spatial distribution of biometals at image resolutions approaching the subcellular level. Ten days after rAAV9-ATP7A administration, copper levels in cerebral cortex, caudate and choroid plexus in the lateral ventricle in combination treated mice were all normal or slightly higher than normal compared to wild type mice and untreated mutants. Immunohistochemistry demonstrated robust ATP7A expression in choroid plexus epithelia as well as in neurons throughout the brain in the treated animals. These preliminary data support the hypothesis that choroid plexus is the major mediator of brain copper delivery by pumping copper into the CSF via ATP7A. Our findings provide further support for CSF-directed viral gene therapy in human subjects with Menkes Disease.
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tandem duplication of exons 1 7 neither impairs atp7a expression nor causes a Menkes Disease phenotype
JIMD reports, 2015Co-Authors: Eun-young Choi, Courtney Holmes, David S Goldstein, Marie Reine Haddad, Keyur Patel, Amalia Dutra, Evgenia Pak, Stephen G KalerAbstract:ATP7A duplications are estimated to represent the molecular cause of Menkes Disease in 4–10% of affected patients. We identified a novel duplication of ATP7A exons 1–7 discovered in the context of a challenging prenatal diagnostic situation. All other reported ATP7A duplications (n = 24) involved intragenic tandem duplications, predicted to disrupt the normal translational reading frame and produce nonfunctional ATP7A proteins. In contrast, the exon 1–7 duplication occurred at the 5′ end of the ATP7A gene rather than within the gene and did not correspond to any known copy number variants. We hypothesized that, if the exon 1–7 duplication was in tandem, functional ATP7A molecules could be generated depending on promoter selection, mRNA splicing, and the proximal and distal duplication breakpoints and that Menkes Disease would be averted. Here, we present detailed molecular characterization of this novel duplication, as well as 2-year postnatal clinical and biochemical correlations. The case highlights the ongoing need for cautious interpretation of prenatal genetic test results.
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in utero copper treatment for Menkes Disease associated with a severe atp7a mutation
Molecular Genetics and Metabolism, 2012Co-Authors: Marie Reine Haddad, Courtney Holmes, David S Goldstein, Charles J Macri, Beryl E Jacobson, Jose A Centeno, Edwina J Popek, W Gahl, Stephen G KalerAbstract: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 (Q724H), 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.
Martina Ralle - One of the best experts on this subject based on the ideXlab platform.
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cerebrospinal fluid directed raav9 rsatp7a plus subcutaneous copper histidinate advance survival and outcomes in a Menkes Disease mouse model
Molecular therapy. Methods & clinical development, 2018Co-Authors: Marie Reine Haddad, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Jose A Centeno, Eun-young Choi, Patricia M. Zerfas, Diego Martinelli, Martina Ralle, Stephen G KalerAbstract: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.
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Cerebrospinal Fluid-Directed rAAV9-rsATP7A Plus Subcutaneous Copper Histidinate Advance Survival and Outcomes in a Menkes Disease Mouse Model
Elsevier, 2018Co-Authors: Marie Reine Haddad, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Jose A Centeno, Eun-young Choi, Patricia M. Zerfas, Diego Martinelli, Martina RalleAbstract: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. Keywords: adeno-associated virus, Menkes Disease, ATP7A, copper, dopamine-beta-hydroxylase, choroid plexus epitheli
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356 high resolution x ray fluorescence microscopy xfm indicates enhanced brain copper delivery in aav9 treated Menkes Disease mice
Molecular Therapy, 2016Co-Authors: Marie Reine Haddad, Patricia M. Zerfas, Martina Ralle, David Vine, Stephen G KalerAbstract:Menkes Disease is a lethal infantile neurodegenerative disorder caused by mutations in a copper transporter, ATP7A. The mottled-brindled (mo-br) mouse recapitulates the symptoms and early demise (14 days) of untreated human Menkes Disease (3 years). We previously demonstrated that viral gene therapy using cerebrospinal fluid (CSF)-directed AAV9-ATP7A in combination with subcutaneous (sc) clinical grade copper histidinate enhanced survival, normalized growth, and improved biochemical and neurobehavioral outcomes in this model. In the current study, we examined copper biodistribution and concentration in specific brain regions for treated and untreated mutants compared to wild type mice, using X-ray fluorescence microscopy (XFM). XFM enables sensitive, quantitative measurement of the spatial distribution of biometals at image resolutions approaching the subcellular level. Ten days after rAAV9-ATP7A administration, copper levels in cerebral cortex, caudate and choroid plexus in the lateral ventricle in combination treated mice were all normal or slightly higher than normal compared to wild type mice and untreated mutants. Immunohistochemistry demonstrated robust ATP7A expression in choroid plexus epithelia as well as in neurons throughout the brain in the treated animals. These preliminary data support the hypothesis that choroid plexus is the major mediator of brain copper delivery by pumping copper into the CSF via ATP7A. Our findings provide further support for CSF-directed viral gene therapy in human subjects with Menkes Disease.
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live cell imaging of compartment specific redox changes in Menkes Disease fibroblasts
Molecular Cytogenetics, 2014Co-Authors: Ashima Bhattacharjee, Martina Ralle, Svetlana LutsenkoAbstract: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.
Anthony Donsante - One of the best experts on this subject based on the ideXlab platform.
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l threo dihydroxyphenylserine corrects neurochemical abnormalities in a Menkes Disease mouse model
Annals of Neurology, 2013Co-Authors: Anthony Donsante, David S Goldstein, Patricia Sullivan, Lauren R Brinster, Stephen G KalerAbstract:Objective Menkes Disease is a lethal neurodegenerative disorder of infancy caused by mutations in a copper-transporting ATPase gene, ATP7A. Among its multiple cellular tasks, ATP7A transfers copper to dopamine-beta-hydroxylase (DBH) within the lumen of the Golgi network or secretory granules, catalyzing the conversion of dopamine to norepinephrine. In a well-established mouse model of Menkes Disease, mottled-brindled, we tested whether systemic administration of L-threo-dihydroxyphenylserine (L-DOPS), a drug used successfully to treat autosomal recessive norepinephrine deficiency, would improve brain neurochemical abnormalities and neuropathology.
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increased frequency of congenital heart defects in Menkes Disease
Clinical Dysmorphology, 2012Co-Authors: Julia D Hicks, Anthony Donsante, Tyler M Pierson, Matthew J Gillespie, Denise E Chou, Stephen G KalerAbstract: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 four of 95 patients 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.
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Favorably skewed X-inactivation accounts for neurological sparing in female carriers of Menkes Disease
Clinical genetics, 2011Co-Authors: Vishal Desai, Anthony Donsante, Kathryn J. Swoboda, M Martensen, Joel A. Thompson, Stephen G KalerAbstract:Classical Menkes Disease is an X-linked recessive neurodegenerative disorder caused by mutations in ATP7A, which is located at Xq13.1-q21. ATP7A encodes a copper-transporting P-type ATPase and plays a critical role in development of the central nervous system. With rare exceptions involving sex chromosome aneuploidy or X-autosome translocations, female carriers of ATP7A mutations are asymptomatic except for subtle hair and skin abnormalities, although the mechanism for this neurological sparing has not been reported. We studied a three-generation family in which a severe ATP7A mutation, a 5.5-kb genomic deletion spanning exons 13 and 14, segregated. The deletion junction fragment was amplified from the proband by long-range polymerase chain reaction and sequenced to characterize the breakpoints. We screened at-risk females in the family for this junction fragment and analyzed their X-inactivation patterns using the human androgen-receptor (HUMARA) gene methylation assay. We detected the junction fragment in the proband, two obligate heterozygotes, and four of six at-risk females. Skewed inactivation of the X chromosome harboring the deletion was noted in all female carriers of the deletion (n = 6), whereas random X-inactivation was observed in all non-carriers (n = 2). Our results formally document one mechanism for neurological sparing in female carriers of ATP7A mutations. Based on review of X-inactivation patterns in female carriers of other X-linked recessive Diseases, our findings imply that substantial expression of a mutant ATP7A at the expense of the normal allele could be associated with neurologic symptoms in female carriers of Menkes Disease and its allelic variants, occipital horn syndrome, and ATP7A-related distal motor neuropathy.
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Molecular correlates of epilepsy in early diagnosed and treated Menkes Disease
Journal of Inherited Metabolic Disease, 2010Co-Authors: Stephen G Kaler, Anthony Donsante, Julia D Hicks, Clarissa J. Liew, Susumu Sato, Jacquelyn C. GreenfieldAbstract: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.
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somatic mosaicism in Menkes Disease suggests choroid plexus mediated copper transport to the developing brain
American Journal of Medical Genetics Part A, 2010Co-Authors: Anthony Donsante, Paul Johnson, Laura A Jansen, Stephen G KalerAbstract: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.
