Stephen G. Kaler, MD, MPH, Head, Unit on Pediatric Genetics
Anthony Donsante, PhD, Postdoctoral Fellow
Sarah Godwin, BA, Postbaccalaureate Fellow¹
Jay Levin, Guest Researcher ²
Jingrong Tang, MD, PhD, Research Fellow

We research problems for which we believe patient-oriented studies can advance understanding 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) platelet biology and hemostasis and (2) 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.

Neonatal diagnosis and treatment of classical Menkes' disease

Kaler, Tang, Godwin, Donsante; in collaboration with Goldstein, Holmes

Menkes' disease is a neurodegenerative disorder of infancy caused by mutations in the copper transport gene ATP7A. Neonatal diagnosis and prompt treatment with copper injections may prevent the mortality and morbidity associated with Menkes' disease; however, early detection is hindered by inadequate sensitivity and specificity of diagnostic tests. We prospectively evaluated the utility of plasma catechol measurements for diagnosing Menkes' disease, assessed the impact of neonatal diagnosis and early treatment on 12 affected newborns, and investigated the molecular basis for disparate clinical outcomes.

Using high-performance liquid chromatography, we measured plasma catechol levels in specimens from 81 at-risk infants referred by hospitals and medical centers throughout the United States (31 states and the District of Columbia) and five foreign countries between May 1997 and July 2005. Twelve of 36 at-risk newborns began treatment with daily copper injections within 10 days of birth. We tracked serum copper, brain MRI, and EEG as markers of medication compliance and brain development. We evaluated survival, growth, and neurodevelopmental function longitudinally for one to eight years. We performed immunohistochemical analyses of patients' fibroblasts by confocal microscopy, yeast complementation assays, and quantitative RT-PCR. The neurochemical patterns of 46 plasma specimens were abnormal, with elevated catechol ratios consistent with decreased DBH activity, while 35 specimens did not show this pattern. Clinical and molecular follow-up confirmed 100 percent sensitivity and specificity of the abnormal catechol pattern for the diagnosis of Menkes' disease. We enrolled 12 affected patients of 10 days of age or less in the NICHD trial of early treatment with copper histidine. Kaplan-Meier estimates of survival were significantly higher among these 12 asymptomatic infants than for a cohort of 12 late-diagnosed symptomatic patients also followed at our institution; three showed good or excellent neurodevelopmental outcomes. Two unrelated treatment responders had the same ATP7A missense mutation, which, as we documented, could complement an S. cerevisiae copper transport mutant (Δccc2). Another responder had a mutation resulting in only 5 percent of the transcript being properly spliced. In contrast, non-responders with large or out-of-frame ATP7A deletions showed no detectable ATP7A protein on confocal microscopy and no functional activity in a yeast complementation assay.

We conclude that plasma catechol measurements are valuable for rapidly diagnosing Menkes' disease, particularly in newborns. Neonatal diagnosis and institution of treatment altered the natural history of the disorder. The most favorable responses to copper histidine occurred in patients whose molecular defects did not entirely abrogate copper transport.

Characterization of pronounced intrafamilial clinical variation in Menkes' disease

Kaler, Donsante, Tang, Godwin; in collaboration with Goldstein, Holmes

Pronounced intrafamilial variability in neurological phenotypes is rare and unusual in Menkes' disease and its variants. We described two unrelated families with several affected individuals with unusually disparate clinical phenotypes and explored the biochemical and molecular underpinnings associated with this variability. Study of intrafamilial variability helps clarify further the molecular bases for what is a rare phenomenon.

Three male siblings, aged two, four, and eight years, had a missense mutation (S637L) previously associated with aberrant mRNA splicing and occipital horn syndrome (Ronce et al., Am J Hum Genet 1997;61:233). The two older siblings showed significant speech and cognitive delays but walked independently beginning at three years of age. The youngest child manifested the severe Menkes' phenotype with global developmental delays. All three brothers had dramatic cerebellar hypoplasia on brain MRI, but significant cortical atrophy was present only in the youngest, who also showed multifocal epileptiform discharges on EEG. The youngest sibling had lower plasma copper levels than the less affected siblings and higher cerebrospinal fluid catechol (CSFC) ratios, the latter suggesting greater deficiency of the copper-dependent enzyme dopamine-β-hydroxylase. Molecular characterization confirmed the presence of some properly spliced transcript and three mutant transcripts in cultured fibroblasts, as noted by Ronce et al., as well as two larger transcripts, one of which retained the whole ATP7A intron 8 (196bp). Quantitative RT-PCR suggested equivalent amounts of properly spliced transcript in all three siblings at levels of about 5 percent of normal; Western analyses using amino-terminal and carboxy-terminal antibodies against ATP7A detected markedly lower quantities of the ATP7A gene product in microsomal preparations of fibroblast protein than in a normal control. Sequencing of copper chaperone genes hCTR1 and Atox1 from genomic DNA showed no differences among the brothers.

In another family, two male siblings age 9 and 19 years had the missense mutation A1362D in exon 21. The older sibling walked independently by two years of age, attended regular school, and communicated effectively without speech or cognitive problems. By contrast, the younger sibling has never walked and shows significant cognitive and speech delays. Brain MRI in the younger sibling at age four showed delayed myelination. He had lower plasma copper levels and higher CSFC ratios than the less affected sibling. RT-PCR of lymphoblast mRNA showed no abnormal splicing, but gene expression in the more severely affected sibling was markedly lower than normal, as determined by RNAse protection assay. We identified no clear evidence for mosaicism in either sibling. Yeast complementation analysis to assess residual copper transport activity for A1362D is in progress.

Adeno-associated virus (AAV) gene therapy in mouse models of Menkes' disease

Kaler, Donsante; in collaboration with Chou, Goldstein, Holmes

Our experience shows that, although mean survival is significantly enhanced, only about 30 percent of patients with Menkes' disease show good or excellent neurological outcomes when treated with copper injections beginning 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 are needed for the large percentage of patients who have little or no residual Atp7a function and currently have no ideal treatment options. Thus, we devised an animal study to assess the efficacy of adeno-associated virus (AAV) gene therapy in mouse models of Menkes' disease.

Gene therapy has been successfully employed to treat a number of genetic disorders in animal models of disease and in at least one human disease. As for the several mouse models of Menkes' disease that now exist, the severity of the disease varies substantially from model to model (as it does from human to human), and not all models respond to copper injections. Thus, when used in combination, the models provide an effective tool for assessing the relationship between disease severity and 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 is responsive to early copper therapy while the other is not. 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 greater fraction of human patients with Menkes' disease.

Hemostasis mediated by the platelet glycoprotein (GP)Ibα-Ibβ-V-IX complex

Tang, Levin, 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: GPIbα, GPIbβ, GPIX, and GPV, which all feature leucine-rich repeat motifs. GPIbβ is arguably the most important component of the complex. A qualitative or quantitative deficiency in this 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 in infancy. Bleeding in BSS is 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 has been characterized), we proposed a new model for the protein structure of GPIbβ. Further study of the protein and its critical role in platelet adhesion and hemostasis is in progress, including efforts to express the protein in a Pichia pastoris gene expression system. A longer-term goal is to generate neutralizing antibodies to GPIbβ for use as anti-thrombotic agents.

PHACES syndrome: X chromosome inactivation and developmental anomalies

Levin, Kaler

The constellation of birth defects that comprises 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. The same situation is seen in PHACES syndrome (posterior fossa brain malformations, hemangiomas, arterial anomalies, coarctation of the aorta and 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 PHACES patient and speculate that the PHACES 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 PHACES phenotype.

¹ Amherst College, Amherst, MA
² Brown University, Providence, RI

COLLABORATORS

Janice Chou, PhD, Heritable Disorders Branch, NICHD, Bethesda, MD
Daniel Goldstein, MD, Clinical Neurosciences Program, NINDS, Bethesda, MD
Courtney Holmes, CMT, Clinical Neurosciences Program, NINDS, Bethesda, MD
Peter Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD

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