Neonatal diagnosis and treatment of Menkes disease

Abstract

Background: Menkes disease is a fatal neurodegenerative disorder of infancy caused by diverse mutations in a copper-transport gene, ATP7A. Early treatment with copper injections may prevent death and illness, but presymptomatic detection is hindered by the inadequate sensitivity and specificity of diagnostic tests. Exploiting the deficiency of a copper enzyme, dopamine-beta-hydroxylase, we prospectively evaluated the diagnostic usefulness of plasma neurochemical levels, assessed the clinical effect of early detection, and investigated the molecular bases for treatment outcomes.

Methods: Between May 1997 and July 2005, we measured plasma dopamine, norepinephrine, dihydroxyphenylacetic acid, and dihydroxyphenylglycol in 81 infants at risk. In 12 newborns who met the eligibility criteria and began copper-replacement therapy within 22 days after birth, we tracked survival and neurodevelopment longitudinally for 1.5 to 8 years. We characterized ATP7A mutations using yeast complementation, reverse-transcriptase-polymerase-chain-reaction analysis, and immunohistochemical analysis.

Results: Of 81 infants at risk, 46 had abnormal neurochemical findings indicating low dopamine-beta-hydroxylase activity. On the basis of longitudinal follow-up, patients were classified as affected or unaffected by Menkes disease, and the neurochemical profiles were shown to have high sensitivity and specificity for detecting disease. Among 12 newborns with positive screening tests who were treated early with copper, survival at a median follow-up of 4.6 years was 92%, as compared with 13% at a median follow-up of 1.8 years for a historical control group of 15 late-diagnosis and late-treatment patients. Two of the 12 patients had normal neurodevelopment and brain myelination; 1 of these patients had a mutation that complemented a Saccharomyces cerevisiae copper-transport mutation, indicating partial ATPase activity, and the other had a mutation that allowed some correct ATP7A splicing.

Conclusions: Neonatal diagnosis of Menkes disease by plasma neurochemical measurements and early treatment with copper may improve clinical outcomes. Affected newborns who have mutations that do not completely abrogate ATP7A function may be especially responsive to early copper treatment. (ClinicalTrials.gov number, NCT00001262.)

Figure 1. Diagnostic Value of Plasma Neurochemicals for Neonatal Detection of Menkes Disease

Panel A is a scatter plot of the ratio of plasma dopamine to norepinephrine versus the ratio of plasma dihydroxyphenylacetic acid to dihydroxyphenylglycol in 36 newborns at risk. Partial deficiency of dopamine-β-hydroxylase in Menkes disease predicts a buildup of proximal metabolites in the normal catecholamine biosynthetic pathway and decreased levels of the distal metabolites. The ratios of dopamine to norepinephrine and of dihydroxyphenylacetic acid to dihydroxyphenylglycol reflect these alterations and distinguish the 14 affected infants from the 22 unaffected infants. The normal pathway of catecholamine biosynthesis is shown below the graph. Panel B shows a receiver-operating-characteristic (ROC) curve for plasma dihydroxyphenylacetic acid in 36 newborns at risk for Menkes disease. ROC curves show the relationship between true positive and false positive rates for a test across various threshold values used to diagnose a condition. The upper curve, plotted by the locally weighted scatter-plot smoothing technique, represents the sensitivity and specificity for the diagnosis of Menkes disease when different cutoff values for dihydroxyphenylacetic acid are applied. The area under the curve (C statistic) for the ROC shown is 0.96. The diagonal line indicates where the curve would rest if a test were completely unreliable (area under the curve, 0.5). The C statistic for plasma dopamine levels in this sample was 1.0 (data not shown).

Figure 2. Neurodevelopmental Outcomes in Patients Who Received a Diagnosis of Menkes Disease as Newborns

Clinical neurodevelopmental outcomes at 36 months of age are shown for Patients 1 to 9 (Panel A). Patient 1 died at 19 months of age. The current neurodevelopmental levels are shown for Patients 10, 11, and 12 at less than 36 months of age (Panel B). The horizontal green lines show the normal level of development for the patient’s age. Del denotes deletion, and ex exon.

Figure 3. Characterization of ATP7A Mutations in an Early-Treatment Cohort of Patients with Menkes Disease

Panel A shows reverse-transcriptase–polymerase-chain-reaction analysis of fibroblast mRNA in a treatment-responsive patient with an IVS9,DS,+6T→G mutation. The patient (II-3 in the pedigree), shown at the age of 32 months, and his older, profoundly disabled brother (II-2), who did not receive an early diagnosis or treatment, both manifested a large quantity of a mutant transcript lacking exon 9, a smaller quantity of the properly spliced 363-bp transcript, and a heteroduplex of both species showing delayed electrophoretic migration. In the pedigree, squares indicate males, circles indicate females, and the diamond indicates four miscarriages. Solid symbols denote affected family members, open symbols unaffected family members, and hatch marks a known carrier of the mutation. In Panel B, a yeast complementation assay distinguishes ATP7A alleles. The plating pattern (clockwise from the 12 o’clock position) includes the yeast copper-transport mutant ccc2 deletion; ccc2 deletion transformed with the normal (wild-type) ATP7A allele; ccc2 deletion mock-transformed with an empty vector; ccc2 deletion transformed with a mutant ATP7A allele harboring deletion of exons 20 to 23; ccc2 deletion transformed with a mutant ATP7A allele, G666R; and ccc2 deletion transformed with the mutant ATP7A allele, N1304S (associated with typical occipital-horn syndrome, a mild variant of Menkes disease, and used as a positive control22). Yeast strains were plated on four different mediums: normal yeast extract–peptone–dextrose (YPD), synthetic yeast nitrogen base (YNB) supplemented with copper (“copper-sufficient” medium), YNB supplemented with iron (“iron-sufficient” medium), and copper- and iron-limited YNB. All strains grew on YPD, copper-sufficient, and iron-sufficient mediums (data not shown), whereas only the wild type, G666R, and N1304S grew on the copper- and iron-limited medium, indicating copper-transport activity associated with these alleles. The wild-type allele shows the most robust growth. The allele with the deletion of exons 20 to 23 failed to complement the knockout strain, indicating no residual copper-transport activity. Panel C shows confocal microscopical images of fibroblasts from a normal male infant and from Patient 2 (deletion of exon 1) stained with a C-terminal antibody to murine ATP7A (red) and the Golgi marker NBD C₆-ceramide (green). In normal control fibroblasts (wild type), there is direct overlap between the ATP7A and Golgi signals (Merge panel), whereas in the cells with the deletion of exon 1, there is no visible ATP7A signal. Del denotes deletion, and ex exon.