Stephen G. Kaler, MD, MPH, Head, Unit on Pediatric Genetics

Po-Ching Liu, DVM, PhD, Research Fellow
Jingrong Tang, MD, PhD, Research Fellow
Sara Stern-Nezer, Guest Researcher*

We focus on several specific areas of biology. Our interests tend to emerge in the context of the infants and children we encounter with certain genetic disorders for which clinical, biochemical, and molecular knowledge is incomplete, for which novel treatment approaches are needed, and for which we believe our patient-oriented studies can be a springboard to advancing understanding in a broader area. Thus, our overarching goal is to improve the understanding, diagnosis, and treatment of these inherited diseases. A longer-term goal is application of genetic approaches to common pediatric diseases for which associated molecular variations will provide the basis both for pathophysiological insights and tailored preventive strategies.

Hemostasis mediated by the platelet glycoprotein (GP)Ibalpha-Ibbeta-V-IX complex

Tang, Liu, Kaler; in collaboration with Steinbach

The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor (vWF) and is composed of the four polypeptides GPIbalpha, GPIbbeta, 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 in infancy. 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 GPIbbeta 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 express human GPIbalpha and GPIX (CHO alphaIX) on their surface only when transfected with wild-type GPIbbeta demonstrated that P96S GPIbbeta 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 GPIbbeta protein structure (see Figure 9.2) that supports the importance of P96 and six other residues previously reported as missense mutations in the conformation of GPIbbeta and its interaction with GPIX. As the most critical component of this recently characterized platelet adhesion complex, GPIbbeta represents an attractive pharmacologic target for modulating hemostasis.
 

Tang J, Stern-Nezer S, Liu P-C, Matyakhina L, Luban N, Kaler SG. A novel mutation in GPIB-beta impairs proper assembly

of the platelet von Willebrand factor receptor and causes Bernard-Soulier syndrome. Am J Hum Genet 2003;73,Suppl 558:2286.

GPIbbeta disorders of copper transport

Liu, Tang, Kaler; in collaboration with Goldstein, Holmes

Menkes' disease is an X-linked recessive neurodegenerative disorder 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. Our work on this disorder includes development of rapid and reliable neurochemical and molecular techniques for very early diagnosis, efforts that dovetail with our clinical trial of very early copper replacement therapy  for affected infants. Our results indicate that the blood-brain barrier poses a challenging obstacle in a majority of patients and suggest a molecular basis for treatment responsivity in the minority of patients with successful neurodevelopmental outcomes. Consequently, we are considering alternative therapeutic approaches that bypass the blood-brain barrier.

To unravel the pathophysiological cascades associated with copper deficiency in the developing brain, we age-matched normal controls (accidental deaths) and performed expression profiling studies on post mortem tissues from several of our patients who died despite very early treatment. The patients' mutations predicted little or no functional copper transport activity. As expected, expression of the Menkes' disease gene was detected in control brains but not in the patient tissue. We detected approximately 350 dysregulated genes, which included those involved in neuronal signaling pathways, synaptic function, mitochondrial function, ribosomal function/translation and metabolism, and a number of ESTs of unknown function. The majority of up-regulated genes were involved in anti-apoptosis and stress response. These findings provided an initial insight into the pathogenetic basis of neurodegeneration in Menkes' disease. We have recently extended our studies to the yeast knock-out strain deltaccc2 that lacks the Menkes/Wilson disease gene homolog to determine those genetic effects of copper deficiency that hold from simple eukaryotes to man.
 

Gasch AT, Caruso RC, Kaler SG, Kaiser-Kupfer M. Menkes' syndrome: ophthalmic findings in Menkes disease.

Ophthalmology 2002;109:1477-1483.

Kaler SG, Holmes CS, Goldstein DS. Perfect sensitivity and specificity of plasma catechol analyses

for neonatal diagnosis of Menkes disease. Pediatr Res 2002;51,225A:1309.

Liu P-C, Chen Y-W, Hoffman EP, Kaler SG. Brain gene expression prole in classical Menkes disease.

Pediatr Res 2002;51,227A:1319.

Liu P-C, Koeller D, Kaler SG. Genomic organization of ATOX1, a human copper chaperone. BMC Genetics

2003;4:4.

Liu P-C, McAndrew PE, Kaler SG. Rapid and robust screening of the Menkes disease/occipital horn

syndrome gene. Genetic Testing 2002;6:255-260.

X chromosome inactivation and developmental anomalies

Tang, Kaler

The constellation of birth defects sternal cleft, abdominal raphe, and hemangiomas shows a distinctive female predilection; available medical literature indicates that nearly all cases (> 92 percent) of this syndrome occur in females. This situation is also seen in the related phenotype PHACE (posterior fossa brain malformations, hemangiomas, arterial anomalies, coarctation of the aorta and cardiac defects and eye abnormalities). 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 PHACE patient and speculate that the phenotype represents an X-linked dominant trait that is lethal in males. We are exploring the hypothesis that defects in a transcription factor or other X chromosomal gene influencing development is responsible for the PHACE phenotype.

Kaler SG, Bochey ME. Skewed X-chromosome inactivation in PHACE syndrome suggests a X-linked

dominant gene. Pediatr Res 2003;53,82A:464.

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 Molecular Modeling, CIT, NIH, Bethesda MD

*Currently at Columbia University, New York NY

For further information, contact kalers@mail.nih.gov