The recently established Unit on Pediatric Genetics investigates several monogenic disorders. Our overarching goal is to improve the understanding, diagnosis, and treatment of these inherited diseases. A longer-term goal is the application of genetic approaches to common pediatric diseases; in these cases, associated molecular variations will provide the basis both for pathophysiological insights and tailored preventive strategies.

Disorders of Copper Transport
Liu, Kaler; in collaboration with Goldstein, Holmes, Hoffman, Chen
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; it also mediates copper exodus from cells. Our work on the disease includes the development of rapid and reliable neurochemical and molecular techniques for very early diagnosis. These efforts dovetail with our clinical trial of very early copper replacement therapy for affected infants. For many years, researchers in the field presumed that early identification and treatment with copper histidine were the key elements in successful management of Menkes’ disease. However, the cumulative clinical, biochemical, and molecular findings from our study indicate that such is not true. Our results show 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 developing alternative therapeutic approaches (e.g., intrathecal copper injections).

To unravel the pathophysiological cascades associated with copper deficiency in the developing brain, we performed Affymetrix (oligonucleotide microarray) expression profiling studies utilizing postmortem tissues from several of our patients who died despite very early treatment. Their 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, including those involved in neuronal signalling pathways, synaptic function, mitochondrial function, and ribosomal function/translation and metabolism, and several ESTs of unknown function. A large share of up-regulated genes was involved in anti-apoptosis and stress response. These findings provide initial insight into the pathogenetic basis of neurodegeneration in Menkes patients with poor response to very early treatment with subcutaneous copper histidine injections.

Molecular Basis of Bernard-Soulier Syndrome
Kaler, in collaboration with Stern-Nezer
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. A qualitative or quantitative deficiency in the complex causes the human platelet disorder Bernard-Soulier syndrome (BSS). Typically, BSS is an autosomal recessive disorder presenting with mild thrombocytopenia, circulating “giant” platelets, and a bleeding phenotype in infancy. 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 at the GP 1b beta locus in an infant haplo-insufficient for the gene as a consequence of heterozygous deletion of chromosome 22q11 (velocardiofacial syndrome). Family studies and functional characterization of the mutant allele are under way with chinese hamster ovary (CHO) cells transfected with the GPIba and GP IX genes. The primary investigations provide the basis for a new clinical protocol to evaluate other individuals with velocardiofacial syndrome (a relatively common genetic syndrome occurring in 1 out of 4,000 live births) for related abnormal-ities in platelet function.

X Chromosome Inactivation and Develop-mental Anomalies
Bochey, Kaler
Nonrandom (or “skewed”) X-chromosome inactivation has been implicated in the etiology of certain X-linked dominant traits (e.g., Rett syndrome). In such situations, female carriers of deleterious alleles on one X chromosome are spared disease manifestations due to favorably skewed X inactivation patterns; their female offspring (in whom X inactivation is random), however, are at risk for expression of the mutant allele. Prenatal lethality in male offspring who inherit the mutant allele explains the observed female predominance.

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