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Translational Research on Inherited Pediatric Diseases

Dr. Stephen G. Kaler, MD, MPH
  • Stephen G. Kaler, MD, MPH, Head, Unit on Pediatric Genetics
  • Jingrong Tang, MD, PhD, Research Fellow
  • Anthony Donsante, PhD, Postdoctoral Fellow
  • Vishal Dilip Desai, BS, Postbaccalaureate Fellow

We research problems for which we believe patient-oriented studies can provide a springboard to advancing understanding in a broader area and for which novel treatment approaches are needed. Our overarching goal is to improve the understanding, diagnosis, and treatment of inherited pediatric diseases. We focus heavily on Menkes disease and occipital horn syndrome, allelic X-linked recessive disorders of copper transport, and maintain interest in two other areas: (1) platelet biology and hemostasis and (2) the clinical and genetic delineation of PHACES, a syndrome of midline developmental defects with strong female predilection. All three projects are associated with clinical conditions that affect infants and children and for which clinical, biochemical, or molecular knowledge is incomplete.

Disorders of copper transport

Desai, Donsante, Tang, Kaler; in collaboration with Goldstein, Holmes, Liew, Patronas, Sato, Steinbach

Menkes disease is an X-linked recessive disorder of copper transport caused by defects in a gene that encodes an evolutionarily conserved copper-transporting ATPase. In mammals, this gene product functions as an intracellular pump to transport copper into trans-Golgi spaces for incorporation into copper-requiring enzymes; it also mediates copper exodus from cells. The disorder presents in infancy with delayed development, failure to thrive, neurodegeneration, and premature death (typically by age 3). Our work on this disorder includes development of rapid and reliable neurochemical and molecular techniques for very early diagnosis, efforts that dovetail with a clinical trial of very early copper histidine treatment for affected infants. We use cell-biological, molecular, and biochemical approaches to characterize enrolled patients and to correlate the results with neurodevelopmental outcomes. We assess quantity and localization of mutant Menkes gene products with confocal imaging of patient fibroblasts. The blood-brain barrier poses a challenging treatment obstacle in many Menkes disease patients, and we hypothesized a molecular basis for treatment responsivity in the minority of patients (about one in five) who respond successfully (normal neurodevelopmental outcomes) to early copper histidine. These patients have mutations that permit at least some residual copper transport to the developing brain. Consequently, we are developing alternative therapeutic approaches, including gene therapy, that bypass the blood-brain barrier.

As above, although mean survival is significantly enhanced, only about 30 percent of patients with Menkes disease show good or excellent neurological outcomes when treated with copper injections beginning very early in life. The main reason for the disparate outcomes appears to be the amount of residual Atp7a function in these individuals. Those with less Atp7a function are unlikely to respond optimally to copper injection treatment. Therefore, new therapeutic strategies are need for the large percentage of patients who have little or no residual Atp7a function and currently have no ideal treatment options.

In a murine model of classical Menkes disease (Atp7aMo-br), which cannot be rescued by intraperitoneal or intravenous copper, we are studying two brain-directed therapies: (1) early intracerebroventricular (IC V) injection of copper chloride and (2) early IC V injection of a recombinant adeno-associated virus serotype 5 (AAV5) vector expressing rsATP7A-AAV, a reduced-size human ATP7A. We developed a genotyping assay that identifies mutant pups before clinical signs appear and administered 50 ng copper chloride to affected mice by ICV injection within three days of birth. We extrapolated the dose from a maximum tolerated dose previously determined in adult rats (Lem et al., Molec Genet Metab 2007;91:30). Treatment modestly extended survival in mutant males and increased mean brain cortex copper levels.

In an alternative approach, we are studying the expression and effects of rsATP7A-AAV delivered by ICV injection. The transgene product begins at methionine 461 of the Menkes ATP7A and includes the fifth and sixth copper-binding domains. In mice receiving 5×108 particles of vector, we documented preliminary evidence of improved brain catechol ratios, reflecting enhanced activity of dopamine-beta-hydroxylase, a copper-dependent enzyme.

Our preliminary data suggest that intracerebroventricular delivery of copper and rsATP7A-AAV results in distribution throughout the Atp7aMo-br brain. Whereas both treatments appeared to improve catecholamine biosynthesis in mutant brains, the effect of copper was more dramatic. Therefore, we may need to refine doses or employ combination therapy in our mouse model to optimize biochemical and clinical outcomes. Brain-directed therapies may ultimately provide a useful approach for treatment of Menkes patients with mutations that cause severe phenotypes.

  • Donsante A, Tang JR, Godwin SC, Holmes CS, Goldstein DS, Bassuk A, Kaler SG. Differences in ATP7A gene expression underlie intra-familial variability in Menkes disease/occipital horn syndrome. J Med Genet 2007;44:492-497.
  • Kaler SG. Wilson disease. In: Goldman L, Ausiello D, eds. Cecil Medicine, Chapter 230. 23rd edition. Saunders Elsevier, 2007.
  • Kaler SG, Holmes CS, Goldstein DS, Tang JR, Godwin SC, Donsante A, Liew CJ, Sato S, Patronas N. Neonatal diagnosis and treatment of Menkes disease. N Engl J Med 2008;358:605-614.
  • Price D, Ravindranath T, Kaler SG. Internal jugular phlebectasia in Menkes disease. Int J Pediatr Otorhinolaryngol 2007;71:1145-1148.
  • Tang J, Donsante A, Desai V, Patronas N, Kaler SG. Clinical outcomes in Menkes disease patients with a copper-responsive ATP7A mutation, G727R. Molec Genet Metab 2008;95:174-181.

Hemostasis mediated by the platelet glycoprotein (GP)Ib alpha-Ib beta-IX complex

Kaler, Tang, Donsante; in collaboration with Steinbach

The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four polypeptides: GPIb alpha, GPIb beta, GPIX, and GPV, which all feature leucine-rich repeat motifs. A qualitative or quantitative deficiency in this complex causes the rare human bleeding diathesis Bernard-Soulier syndrome (BSS). BSS is an autosomal recessive trait presenting in infancy with thrombocytopenia, circulating “giant” platelets, and bleeding tendency. Bleeding in BSS is more severe than predicted by platelet count and is explained by a defect in primary hemostasis. We identified a novel mutation (P96S) at the GPIb beta locus in an infant haploinsufficient for this gene, with the haploinsufficiency attributable to heterozygous deletion of chromosome 22q11 (velocardiofacial syndrome). Using flow cytometry and confocal imaging of transfected Chinese hamster ovary cells that stably surface-express human GPIb alpha and GPIX (CHO alpha-IX) when transfected with wild-type GPIb beta, we demonstrated that P96S GPIb beta abrogates surface assembly of the platelet vWF receptor complex. Based on amino acid homology to the nogo-66 neuronal receptor (also a leucine-rich repeat protein, whose crystal structure has been characterized), we proposed a model of GPIb beta protein structure that both supports the importance of P96 and other residues previously reported as mis-sense mutations in the conformation of GPIb beta and demonstrates P96’s interaction with GPIX. GPIb beta represents the most crucial component of this recently characterized platelet adhesion complex. Further study of GPIb beta and its critical role in platelet adhesion and hemostasis is in progress to illuminate more precisely GPIb beta’s interaction with GPIX and GPIb alpha and to develop novel therapeutic approaches for BSS patients. Our current efforts involve expression of a FLAG-tagged GPIb beta cDNA in HEK-293 cells in order to purify the native protein for crystallographic analysis and generate specific antibodies against it for future studies of the (GP)Ib-V-IX complex.

Clinical and molecular characterization of PHACES syndrome


The acronym PHACES describes the association of posterior fossa malformations, hemangiomas, arterial anomalies (cardiovascular or cerebrovascular), coarctation of the aorta and cardiac defects, eye abnormalities, and sternal or ventral defects. We studied a female patient with an uncommon variant of this neurocutaneous disorder who manifested micrognathia; hemangiomas of the face, chest, and extremities; cerebrovascular anomalies; supra-umbilical raphe; and sternal cleft. A literature review of PHACES patients with both supra-umbilical raphe and sternal cleft revealed a marked female predilection. Taken together with cases of facial hemangiomas, supra-umbilical raphe, and sternal cleft reported in the literature, 91 percent (40/44) of patients are female. One affected male died shortly after birth. We hypothesized that the gender bias in PHACES results from mutation in an X-linked dominant gene that is often lethal in males; we performed X-inactivation analysis of the polymorphic androgen receptor locus in the family of the affected infant. We documented consistently skewed X-inactivation (80 percent/20 percent in two independent analyses) in the unaffected mother and consistently random X-inactivation (47:53 and 61:39 in independent analyses) in the proband. Our findings are consistent with favorably skewed X-inactivation producing a normal maternal phenotype, a phenomenon documented in X-linked dominant Rett syndrome. In searching for submicroscopic copy number changes in PHACES patients, our future efforts will depend on (1) ascertainment of other PHACES families in which maternal X-inactivation studies may be pursued and (2) application of X-chromosome–specific array-comparative genomic hybridization (array-CGH) experiments.

  • Levin JH, Kaler SG. Nonrandom maternal X chromosome inactivation associated with PHACES. Clin Genet 2007;72:345-350.


  • David S. Goldstein, MD, PhD, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • Courtney Holmes, CMT, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • Clarissa Jiang Liew, MD, Epilepsy Research Branch, NINDS, Bethesda, MD
  • Nicholas Patronas, MD, Diagnostic Radiology Department, Clinical Center, NIH, Bethesda, MD
  • Susumu Sato, MD, Epilepsy Research Branch, NINDS, Bethesda, MD
  • Peter Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD

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