BRIDGING THE GAP BETWEEN RARE DISEASES AND COMPLEX
INHERITED DISORDERS OF CHILDHOOD
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Stephen G. Kaler, MD, MPH, Head, Unit on Pediatric Genetics Po-Ching Liu,
DVM, PhD, Research Fellow Kristen Lem,
AB, Guest Researchera |
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The
Unit on Pediatric Genetics is interested in several specific areas of
biology; these interests emerge in the context of infants and children with
genetic disorders for which clinical, biochemical, and molecular knowledge is
incomplete; for which novel treatment approaches are needed; and from which
patient-oriented studies can advance understanding in a broader area. Our overarching goal is to improve the understanding,
diagnosis, and treatment of rare inherited pediatric diseases. A longer-term
goal is to apply genetic approaches to common pediatric diseases for which
associated molecular variations will provide the basis for both
pathophysiological insights and tailored preventive strategies. 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 (WF)
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 disproportionately 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 the gene as a consequence of
heterozygous deletion of chromosome 22q11 (velocardiofacial syndrome).
We
used flow cytometry and confocal imaging (Figure 9.2) of transfected
Chinese hamster ovary cells that stably surface-express human GPIb-alpha and GPIX (CHOalphaIX)
when transfected with wild-type GPIb-beta
to demonstrate 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, the crystal structure of which has been
characterized), we proposed a model of GPIb-beta
protein structure that supports the importance of P96 and other residues
previously reported as missense mutations in the conformation of GPIb-beta and its 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 with the goal of developing novel therapeutic
approaches for BSS patients, illuminating more precisely GPIb-beta’s
interaction with GPIX and GPIb-alpha, and
identifying GPIb-beta as potential target for
anti-thrombotic drug development. Tang
J, Liu PC, Steinbach PJ, Luban NLC, Kaler SG. Expression
studies and homology modeling of GPIb beta. Pediatr Res 2004;55:271A/1543. 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 Ib-beta
impairs assembly of von Willebrand factor receptor. Throm Haemost
2004;92:75-88. Disorders of copper
transport Lem, Liu, Tang, Kaler; in collaboration with Goldstein, Holmes 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 and mediates copper exodus from cells. The disorder presents in
infancy with delayed development, failure to thrive, neurodegeneration,
and premature death (typically by three years of age). 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 their neurodevelopmental outcomes. We rely on confocal
imaging of patient fibroblasts to assess quantity and localization of mutant
Menkes gene products. The blood-brain barrier poses a challenging obstacle in
many Menkes disease patients, and we proposed a molecular basis for treatment
responsivity in the minority of patients (about one
in five) who respond (normal neurodevelopmental outcomes) to early copper
histidine. These patients have mutations that allow at least some residual
copper transport to the developing brain. Consequently, we are developing
alternative therapeutic approaches, including intrathecal copper administration, that bypass the blood-brain barrier.
To assess safety and to
determine a maximum tolerated dose (MTD), we began an animal protocol of
intraventricular copper histidine using adult male rats and established a
maximum tolerated dose of 5 micrograms. Two weeks post-injection, some
inflammatory changes are evident in the periventricular region regardless of
dose (Figure 9.3), but they do not appear to be clinically significant.
Studies of chronic administration (i.e., weekly administration) of the MTD
are in progress. A small study of intrathecal copper administration in
primates may also be warranted before beginning human trials. Kaler SG. ATP7A-related copper transport disorders. In: GeneReviews at
GeneTests: Medical Genetics Information Resource
[online database]. Copyright, Kaler SG. Menkes disease. In Robertson D, Low PA, Burnstock G, Biaggioni I, eds. Primer on the
Autonomic Nervous System. 2nd Edition. Chapter 74. Kaler SG. Kaler SG, Liu P-C, Tang JR, Lem KE. Response to very early copper treatment in
classical Menkes disease. Am J Hum Genet, in
press. Liu P-C, Koeller D, Kaler SG. Genomic organization of ATOX1, a human copper
chaperon. BMC Genet 2003;4:4. X chromosome
inactivation and developmental anomalies Tang, Kaler The constellation of birth
defects including sternal cleft, abdominal raphe, and hemangiomas shows a
distinctive female predilection; available medical literature indicates that
nearly all (over 92 percent) cases 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
because of 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 this
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 an X-linked dominant
gene. Pediatr Res 2003;53:82A/464. COLLABORATORS David S.
Goldstein, MD, Clinical Neurosciences
Program, NINDS, Courtney S. Holmes, CMT,
Clinical Neurosciences Program, NINDS, Peter J. Steinbach, PhD, Center
for Information Technology, NIH, For further information, contact kalers@mail.nih.gov |
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