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Stephen Kaler, MD, Head, Unit on Pediatric Genetics
Jingrong Tang, MD, PhD, Research Fellow
Anthony Donsante, PhD, Postdoctoral Fellow
Vishal Dilip Desai, BS, Postbaccalaureate Fellow
Sarah C. Godwin, BA, Postbaccalaureate Fellow1

Photo of Stephen Kaler, M. D.

We research problems for which we believe patient-oriented studies can advance knowledge 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. In the past year, we focused heavily on Menkes disease, an X-linked recessive disorder of copper transport, and continued some clinical and laboratory work in two other areas: (1) the clinical and genetic delineation of PHACES, a syndrome of midline developmental defects with strong female predilection, and (2) platelet biology and hemostasis. 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

Our goals are to improve the diagnosis and treatment of human disorders of copper transport, including Menkes and Wilson diseases, and the occipital horn syndrome. Taking a “bedside to bench” approach, we also seek to identify and characterize underappreciated cellular and genetic phenomena suggested by patients’ phenotypes or their responses to treatment.

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

Even though their mean survival is significantly enhanced, only 30 percent of patients with Menkes disease show good or excellent neurological outcomes when treated with copper injections that begin 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 need to be developed for the large percentage of patients who have little or no residual Atp7a function and thus no ideal treatment options. Toward this goal, we proposed an animal study, recently approved, to assess the efficacy of adeno-associated virus (AAV) gene therapy in mouse models of Menkes disease.

Gene therapy offers an alternative treatment for genetic disorders, such as Menkes disease, that result from the loss of a protein function. It provides a way to restore the protein function in an affected individual and has been successfully employed to treat several genetic disorders in animal models of disease and in at least one human disease. Several mouse models of Menkes disease exist. As with human patients, the severity of the disease varies substantially from model to model, and only some models respond to copper injections. Thus, when used in combination, the models provide an effective tool for evaluating the effects of disease severity on the effectiveness of novel treatments. The goal of our study is to evaluate the use of recombinant adeno-associated virus serotype 5 (rAAV5) as a gene therapy vector in two mouse models of Menkes disease, one responsive and the other unresponsive to early copper therapy. We will evaluate efficacy by examining life span and several biochemical parameters that are abnormal in Menkes disease mice and Menkes disease patients. If successful, the experiments will lay the groundwork for more effective therapies for a higher percentage of human patients with Menkes disease.

Donsante A, Tang J, Godwin SC, Holmes CS, Goldstein DS, Bassuk A, Kaler SG. Differences in ATP7A gene expression underlie intrafamilial variability in Menkes disease/occipital horn syndrome. J Med Genet 2007;44:492-7.

Godwin SC, Shawker T, Chang B, Kaler SG. Brachial artery aneurysms in Menkes disease. J Pediatr 2006;149:412-5.

Lem KE, Brinster LR, Tjurmina O, Lizak M, Lal S, Centeno JA, Liu PC, Godwin SC, Kaler SG. Safety of intracerebroventricular copper histidine in adult rats. Mol Genet Metab 2007;91:30-6.

Price DJ, Ravindranath T, Kaler SG. Internal jugular phlebectasia in Menkes disease. Int J Pediatr Otorhinolaryngol 2007;71:1145-8.

Tang J, Robertson S, Lem KE, Godwin SC, Kaler SG. Functional copper transport explains neurologic sparing in occipital horn syndrome. Genet Med 2006;8:711-8.

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; she manifested a sternal cleft; supraumbilical raphe; hemangiomas of the face, chest, and extremities; micrognathia; and cerebrovascular anomalies. A literature review of PHACES patients with both sternal cleft and supraumbilical raphe revealed a marked female predilection. Taken together with cases of sternal cleft, supraumbilical raphe, and facial hemangiomas compiled in the literature, we observed that 91 percent (40/44) of patients are female. One affected male died shortly after birth. Hypothesizing 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 proband. We documented consistently skewed X-inactivation (80 and 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. The findings are consistent with favorably skewed X-inactivation producing a normal maternal phenotype, a phenomenon documented in X-linked dominant Rett syndrome. Our future efforts will depend on ascertainment of other PHACES families in which maternal X-inactivation studies can be pursued as well as on application of X-chromosome–specific array–comparative genomic hybridization (array–CGH) experiments to search for submicroscopic copy number changes in PHACES syndrome patients. These objectives may involve a new natural history protocol to recruit PHACES patients and families to the NIH Clinical Center.

Levin JH, Kaler SG. Nonrandom maternal X chromosome inactivation associated with PHACES. Clin Genet 2007;72:345-50.

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

The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor (vWF) and is composed of four polypeptides: GPIb alpha, GPIb beta, GPIX, and GPV, all featuring 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 would be 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 the gene as a consequence of heterozygous deletion of chromosome 22q11 (velocardiofacial syndrome). 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 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 supports the importance of P96 and other residues—previously reported as mis-sense mutations in the conformation of GPIb beta—and P96’s interaction with GPIX. GPIb beta represents the most important 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 its interaction with GPIX and GPIb alpha, with the hope of developing 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 to generate specific antibodies against the protein for future studies of the (GP)Ib-V-IX complex.

1 Amherst College, Amherst, MA


Daniel Goldstein, MD, 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, NIH Clinical Center, Bethesda, MD
Susumu Sato, MD, Epilepsy Research Branch, NINDS, Bethesda, MD
Peter Steinbach, PhD, Center for Molecular Modeling, NIH, Bethesda, MD

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