Our overarching goal is to improve the understanding, diagnosis, and treatment of inherited pediatric diseases. In the past year, we focused on Menkes disease, an X-linked recessive disorder of copper transport, while extending our work in two other areas: platelet biology and hemostasis and 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
Godwin, Lem, Levine, Liu, Tang, Kaler; in collaboration with Goldstein, Holmes
Menkes disease (MD) 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, the gene product functions as an intracellular pump to transport copper into trans-Golgi spaces for incorporation into copper-requiring enzymes and to mediate copper exodus from cells. MD presents in infancy with delayed development, failure to thrive, neurodegeneration, and premature death (typically by three years of age).
We used robust neurochemical and molecular methods for very early diagnosis of MD, facilitating recruitment of patients to our clinical trial of very early copper histidine treatment for affected infants. Neonatal diagnosis and prompt treatment with copper injections can prevent the mortality and morbidity associated with MD; however, inadequate sensitivity and specificity of commonly available biochemical tests hinder early detection. Thus, we evaluated the sensitivity and specificity of plasma neurochemical measurements as a diagnostic test for MD in 81 at-risk male infants referred by hospitals and medical centers throughout the United States (31 states and the District of Columbia) and in five foreign countries between May 1997 and April 2005. MD patients are characterized by abnormal neurochemical levels related to partial deficiency of the copper-containing enzyme dopamine-beta-hydroxylase (DBH). Of the 81 specimens referred to us, 46 had an abnormal neurochemical pattern consistent with decreased DBH activity, and 35 did not exhibit the pattern. Among a subgroup of 36 asymptomatic newborns with a family history of MD, 14 showed the abnormal pattern and 22 did not. Clinical and molecular follow-up confirmed 100 percent sensitivity and 100 percent specificity of the abnormal catechol pattern among the entire cohort. Thirty percent of asymptomatic affected infants identified and treated within 10 days of birth had highly favorable clinical outcomes. We conclude that plasma catechol measurements are sensitive and specific for diagnosing MD, particularly in newborn infants for whom very early recognition is a prerequisite for successful therapeutic intervention in this otherwise lethal disorder. Measurement of plasma neurochemicals by tandem mass spectroscopy may be useful in efforts to establish newborn screening for MD.
The blood-brain barrier poses a challenging obstacle in MD patients whose mutations do not permit residual copper transport to the developing brain. Consequently, we are exploring alternative therapeutic approaches, particularly intracerebroventricular (ICV) copper administration, that bypass the blood-brain barrier. Several important issues are related to the potential efficacy of this approach. First, strong experimental evidence suggests the necessity for the expression of high-affinity copper uptake genes (e.g., hCTR1) and copper chaperones (e.g., Cox17, CCS1) in human neuronal cells. A second requirement is that the distribution and cerebrospinal fluid clearance of copper administered by ICV injection favor neuronal uptake. In examples of other trace metals, e.g., zinc chloride (65ZnCl2), zinc histidine (65Zn-His), and manganese chloride (55MnCl2), autoradiography or magnetic resonance imaging following ICV injection has documented wide distribution and sustained presence within brain. The results augur well for neuronal delivery of copper. In the case of small molecules, e.g., atomic mass of 55 Da for manganese, 63.5 Da for copper, and 65.4 Da for zinc, the distribution and brain penetration of trace metal ions, especially paramagnetic ions such as Mn and Cu, administered by ICV injection are not comparable to the distribution and penetration of larger drug molecules that have been shown to have limited brain penetration.
To evaluate safety and determine a maximum tolerated dose (MTD) of copper histidine by ICV administration, we performed an animal protocol in adult male rats and established an MTD of 0.5 microgram. The dose was associated with normal growth and behavior over a four-week period, although some local inflammatory and other histopathological brain changes were evident in some animals. Further study of ICV copper histidine administration in primates may be warranted before human trials.
Grange DK, Kaler SG, Albers GM, Petterchak JA, Thorpe CM, deMello DE. Severe bilateral panlobular emphysema and pulmonary arterial hypoplasia: unusual manifestations of Menkes disease. Am J Med Genet A 2005;139:151-155.
Kaler SG. ATP7A-related copper transport disorders. In: GeneReviews at GeneTests: Medical Genetics Information Resource [database online]. Copyright, University of Washington, Seattle, 1997-2005. May 2005, available at http://www.genetests.org.
Kaler SG. Wilson disease. In: Goldman L, Ausiello D, eds. Cecil’s Textbook of Medicine. 23rd edition. Philadelphia: Saunders (in press).
Liu PC, Chen YW, Centano J, Quesado M, Lem KE, Kaler SG. Downregulation of myelination, energy, and translational genes in Menkes disease brain. Mol Genet Metab 2005;85:291-300.
Sheela SR, Manoj L, Liu P-C, Lem KE, Kaler SG. Copper replacement treatment for symptomatic Menkes disease: ethical considerations. Clin Genet 2005;68:278-283.
Hemostasis mediated by the platelet glycoprotein–Ib-alpha-Ib-beta-V-IX complex
Tang, Lem, Liu, Kaler; 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; GPIb-beta is arguably the most important component of the complex. A qualitative or quantitative deficiency in the complex causes Bernard-Soulier syndrome (BSS), a human bleeding diathesis. BSS is an autosomal recessive trait that presents in infancy with thrombocytopenia, circulating “giant” platelets, and bleeding tendency during infancy. Bleeding in BSS is disproportionately more severe than predicted by platelet count and is explained by a defect in primary hemostasis. Based on amino acid homology to the nogo-66 neuronal receptor (also a leucine-rich repeat protein, whose crystal structure is known), we proposed a new model for the protein structure of GPIb-beta. Further study of the protein and its critical role in platelet adhesion and hemostasis is in progress. A long-term goal is to generate neutralizing antibodies to GPIb-beta for use as anti-thrombotic agents.
Kaler SG. Genetic disorders of the platelet glycoprotein Ib-IX complex. Mol Med (in press).
Tang J, Stern-Nezer S, Liu PC, Matyakhina L, Riordan M, Luban NCL, Steinbach PJ, Kaler SG. Mutation in the leucine-rich repeat C-flanking region of platelet glycoprotein Ibb impairs assembly of von Willebrand factor receptor. Thromb Haemost 2004;92:75-88.
PHACE syndrome: X-chromosome inactivation and developmental anomalies
Levin, Tang, Kaler
The constellation of birth defects that includes sternal cleft, abdominal raphe, and hemangiomas shows a distinctive female predilection; the available medical literature indicates that nearly all cases (over 92 percent) of the syndrome occur in females. The same holds true for PHACE syndrome (posterior fossa brain malformations, hemangiomas, arterial anomalies, coarctation of the aorta, cardiac defects, and eye abnormalities), which may represent an allelic variant. Nonrandom (or “skewed”) X-chromosome inactivation has been implicated in the etiology of certain X-linked dominant traits. In such situations, female carriers of deleterious alleles on one X chromosome are spared disease manifestations due to favorably skewed X-inactivation patterns; however, their female offspring (in whom X inactivation is random) are at risk for expression of the mutant allele. Prenatal lethality in male offspring who inherit the mutant allele explains the observed female predominance. We documented skewed X inactivation in the mother of a female PHACE syndrome patient and speculate that the PHACE syndrome phenotype represents an X-linked dominant trait that is lethal in males. We are exploring the hypothesis that defects in an X-chromosomal gene influencing development is responsible for the PHACE syndrome phenotype.
Kaler SG, Bochey ME. Skewed X-chromosome inactivation in PHACE syndrome suggests an X-linked dominant gene. Pediatr Res 2003;53 Part 2:82A/464.
Levin JH, Kaler SG. Sternal cleft, supraumbilical raphe, and hemangiomas: a rare variant of PHACE syndrome. Clin Genet (in press).
1Amherst College, Amherst, MA
2Brown University, Providence, RI
Collaborators
David S. Goldstein, MD, Clinical Neurosciences Program, NINDS, Bethesda, MD
Courtney S. Holmes, CMT, Clinical Neurosciences Program, NINDS, Bethesda, MD
Peter J. Steinbach, PhD, Center for Information Technology, NIH, Bethesda, MD
For further information, contact kalers@mail.nih.gov.