In utero copper treatment for Menkes disease associated with a severe ATP7A mutation.

Menkes disease is a lethal X-linked recessive neurodegenerative disorder of copper transport caused by mutations in ATP7A, which encodes a copper-transporting ATPase. Early postnatal treatment with copper injections often improves clinical outcomes in affected infants. While Menkes disease newborns appear normal neurologically, analyses of fetal tissues including placenta indicate abnormal copper distribution and suggest a prenatal onset of the metal transport defect. In an affected fetus whose parents found termination unacceptable and who understood the associated risks, we began in utero copper histidine treatment at 31.5 weeks gestational age. Copper histidine (900 µg per dose) was administered directly to the fetus by intramuscular injection (fetal quadriceps or gluteus) under ultrasound guidance. Percutaneous umbilical blood sampling enabled serial measurement of fetal copper and ceruloplasmin levels that were used to guide therapy over a four-week period. Fetal copper levels rose from 17 µg/dL prior to treatment to 45 µg/dL, and ceruloplasmin levels from 39 mg/L to 122 mg/L. After pulmonary maturity was confirmed biochemically, the baby was delivered at 35.5 weeks and daily copper histidine therapy (250 µg sc b.i.d.) was begun. Despite this very early intervention with copper, the infant showed hypotonia, developmental delay, and electroencephalographic abnormalities and died of respiratory failure at 5.5 months of age. The patient's ATP7A mutation (Q724H), which severely disrupted mRNA splicing, resulted in complete absence of ATP7A protein on Western blots. These investigations suggest that prenatally initiated copper replacement is inadequate to correct Menkes disease caused by severe loss-of-function mutations, and that postnatal ATP7A gene addition represents a rational approach in such circumstances.

Adrenomedullary response to glucagon in patients with primary Sjögren's syndrome.

Several studies showed signs of autonomic dysfunction in patients with primary Sjögren's syndrome (pSS). Adrenomedullary function might be of importance for pSS pathogenesis by affecting salivary gland functions and modulating immune responses. The aim of the study was to evaluate the adrenomedullary hormonal system in patients with pSS. The glucagon test (1 mg i.v.) was performed in 18 pSS patients and 13 control subjects. During the test each patient had electrocardiographic and impedance cardiographic monitoring. Plasma epinephrine and norepinephrine were assayed by liquid chromatography with electrochemical detection after batch alumina extraction. Baseline concentrations of epinephrine and norepinephrine were comparable between pSS and controls. Glucagon administration induced a significant increase in systolic blood pressure, diastolic blood pressure, heart rate, cardiac output (P < 0.01), and stroke volume; however, the changes were comparable between pSS and controls. Epinephrine levels increased (P < 0.01) in response to glucagon administration while norepinephrine concentration did not change. There was no significant difference in neurochemical responses to glucagon between pSS and controls. In conclusion, the present results suggest normal adrenomedullary function in pSS.

Neonatal diagnosis and treatment of Menkes disease.

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.)

Relative efficiencies of plasma catechol levels and ratios for neonatal diagnosis of menkes disease.

BACKGROUND: Menkes disease is an X-linked recessive neurodevelopmental disorder resulting from mutation in a copper-transporting ATPase gene. Menkes disease can be detected by relatively high concentrations of dopamine (DA) and its metabolites compared to norepinephrine (NE) and its metabolites, presumably because dopamine-beta-hydroxylase (DBH) requires copper as a co-factor. The relative diagnostic efficiencies of levels of catechol analytes, alone or in combination, in neonates at genetic risk of Menkes disease have been unknown. METHODS: Plasma from 44 at-risk neonates less than 30 days old were assayed for DA, NE, and other catechols. Of the 44, 19 were diagnosed subsequently with Menkes disease, and 25 were unaffected. RESULTS: Compared to unaffected at-risk infants, those with Menkes disease had high plasma DA (P < 10(-6)) and low NE (P < 10(-6)) levels. Considered alone, neither DA nor NE levels had perfect sensitivity, whereas the ratio of DA:NE was higher in all affected than in all unaffected subjects (P = 2 x 10(-8)). Analogously, levels of the DA metabolite, dihydroxyphenylacetic acid (DOPAC), and the NE metabolite, dihydroxyphenylglycol (DHPG), were imperfectly sensitive, whereas the DOPAC:DHPG ratio was higher in all affected than in all unaffected subjects (P = 2 x 10(-4)). Plasma dihydroxyphenylalanine (DOPA) and the ratio of epinephrine (EPI):NE levels were higher in affected than in unaffected neonates (P = 0.0015; P = 0.013). CONCLUSIONS: Plasma DA:NE and DOPAC:DHPG ratios are remarkably sensitive and specific for diagnosing Menkes disease in at-risk newborns. Affected newborns also have elevated DOPA and EPI:NE ratios, which decreased DBH activity alone cannot explain.

Differences in ATP7A gene expression underlie intrafamilial variability in Menkes disease/occipital horn syndrome.

BACKGROUND: Pronounced intrafamilial variability is unusual in Menkes disease and its variants. We report two unrelated families featuring affected members with unusually disparate clinical and biochemical phenotypes and explore the underlying molecular mechanisms. METHODS: We measured biochemical markers of impaired copper transport in five patients from two unrelated families and used RNase protection, quantitative reverse transcription (RT)-PCR, Western blot analysis and yeast complementation studies to characterise two ATP7A missense mutations, A1362D and S637L. RESULTS: In two brothers (family A) with A1362D, RNase protection and Western blot analyses revealed higher amounts of ATP7A transcript and protein in the older, mildly affected patient, who also had a higher plasma copper level and lower cerebrospinal fluid dihydroxyphenylalanine : dihydroxyphenylglycol ratio. These findings indicate greater gastrointestinal absorption of copper and higher activity of dopamine-beta-hydroxylase, a copper-dependent enzyme, respectively. In family B, three males with a missense mutation (S637L) in an exon 8 splicing enhancer showed equally reduced amounts of ATP7A transcript and protein by quantitative RT-PCR and western blot analysis, respectively, despite a more severe phenotype in the youngest. This patient's medical history was notable for cardiac arrest as a neonate, to which we attribute his more severe neurodevelopmental outcome. CONCLUSIONS: These families illustrate that genetic and non-genetic mechanisms may underlie intrafamilial variability in Menkes disease and its variants.

Predominant Glandular Cholinergic Dysautonomia in Patients With Primary Sjögren's Syndrome.

OBJECTIVE: The autonomic nervous system (ANS) modulates exocrine gland function. Available data show poor correlation between the degree of function and destruction of the exocrine glands in primary Sjögren's syndrome (SS), suggesting that other mechanisms, such as autonomic dysfunction, may be important in these patients. The aim of this study was to perform a comprehensive analysis of sympathoneural and sympathetic cholinergic function in well-characterized patients with primary SS. METHODS: Twenty-one patients with primary SS (mean ± SEM age 44.2 ± 2.8 years) and 13 healthy control subjects (mean ± SEM age 50.8 ± 1.9 years) were assessed during orthostasis and intravenous injection of edrophonium (10 mg). The postganglionic sympathetic cholinergic system was evaluated by assessing sweat production by means of the Quantitative Sudomotor Axon Reflex Test (QSART). Tests of gastric emptying were used to assess the gastrointestinal ANS in primary SS patients. RESULTS: The velocity index and the acceleration index were significantly higher (P < 0.05) in patients with primary SS as compared to controls, both before and during the orthostatic and edrophonium tests. Findings of other hemodynamic and neurochemical parameters did not differ between primary SS patients and controls during the orthostasis and edrophonium test; however, the edrophonium-induced saliva increment was lower in primary SS patients (P = 0.002). Abnormally low sweat production was found in 4 primary SS patients but in none of the controls, as determined by the QSART. Gastric empting was delayed in 53% of primary SS patients. CONCLUSION: We observed subtle differences in several ANS domains, including the gastrointestinal and sympathocholinergic systems, suggesting the presence of a complex ANS dysfunction in primary SS. The impact was greatest on the exocrine glands, with subtle differences in the cardiac parasympathetic function that were independent of glandular inflammation and atrophy, suggesting an alternative mechanism of disease pathogenesis in primary SS.

Effects of risperidone on the peripheral noradrenegic system in patients with schizophrenia: a comparison with clozapine and placebo.

Risperidone is an atypical antipsychotic drug that increases plasma norepinephrine (NE) levels, but the mechanism behind this effect is unclear. We measured arterial plasma levels of NE and other catechols during intravenous infusion of tritium-labeled NE (3H-NE) in risperidone-treated patients and compared their data with those from patients treated with clozapine or placebo. NE levels in risperidone patients were significantly higher than in placebo patients, but lower than in clozapine patients. Neither drug, however, had significant effect on plasma levels of the main neuronal metabolite of NE, dihydroxyphenylglycol (DHPG), suggesting that adrenoceptors blockade alone would not explain the NE findings. The rate of release of endogenous NE into the bloodstream (spillover) was elevated in both risperidone and clozapine patients in a manner that paralleled their NE levels; the NE clearance in both groups did not differ from placebo. Following 3H-NE infusion in risperidone-treated individuals, production of 3H-DHPG was normal, as it was in the clozapine group, suggesting that risperidone does not impede neuronal uptake or intraneuronal metabolism of NE by monoamine oxidase. Our data suggest that both risperidone and clozapine elevate plasma NE levels via enhanced neurotransmitter spillover, with risperidone producing a smaller effect.

Catecholamine metabolites affected by the copper-dependent enzyme dopamine-beta-hydroxylase provide sensitive biomarkers for early diagnosis of menkes disease and viral-mediated ATP7A gene therapy.

Menkes disease is a lethal X-linked recessive disorder of copper metabolism caused by mutations in ATP7A, a copper-transporting ATPase with diverse and important biological functions. Partial deficiency of dopamine-beta-hydroxylase is a biochemical hallmark of this illness due to the normal role of ATP7A in delivery of copper as an enzymatic cofactor. We exploited this fact to develop a diagnostic test for Menkes disease, which proved highly sensitive and specific. The assay has enabled early identification of affected patients, leading to enhanced survival and improved neurodevelopment after early copper treatment, including some completely normal outcomes. In preclinical efforts to develop improved therapies for patients with non-copper-responsive ATP7A mutations, we used brain-directed adeno-associated viral gene therapy to rescue a murine model of the disease. Statistically significant improvement in brain catechol ratios correlated with enhanced survival, and cerebrospinal fluid catechols represent candidate surrogate markers of treatment effect in a future gene therapy clinical trial.

Diagnosis of copper transport disorders.

Techniques for the diagnosis of copper transport disorders are increasingly important due to recent recognition of previously unappreciated clinical phenotypes and emerging advances in the treatment of these conditions. Here, we collate the diagnostic approaches and techniques currently employed for biochemical and molecular assessment of at-risk individuals in whom abnormal copper metabolism is suspected.

Failure of propranolol to prevent tilt-evoked systemic vasodilatation, adrenaline release and neurocardiogenic syncope.

In patients with neurocardiogenic syncope, head-up tilt often evokes acute loss of consciousness accompanied by vasodilatation, increased plasma adrenaline and systemic hypotension. Since hypotension increases adrenaline levels and adrenaline can produce skeletal muscle vasodilatation by activating beta2 receptors, adrenaline might induce a positive feedback loop precipitating circulatory collapse. We hypothesized that propranolol, a non-selective beta-blocker, would prevent adrenaline-induced vasodilatation and thereby prevent syncope. Eight subjects with recurrent neurocardiogenic syncope and previously documented tilt-induced syncope with elevated plasma adrenaline levels participated in the present study. Subjects underwent tilt table testing after receiving oral propranolol or placebo in a double-blind randomized crossover fashion. Haemodynamic and neurochemical variables were measured using intra-arterial monitoring, impedance cardiography, arterial blood sampling and tracer kinetics of simultaneously infused [3H]noradrenaline and [3H]adrenaline. The occurrence of tilt-induced neurally mediated hypotension and syncope, duration of tilt tolerance, extent of the decrease in SVRI (systemic vascular resistance index) and magnitude of plasma adrenaline increases did not differ between the propranolol and placebo treatment phases. SVRI was inversely associated with fractional increase in plasma adrenaline during both phases. One subject did not faint when on propranolol; this subject's response is discussed in the context of central effects of propranolol. In this small, but tightly controlled, study, propranolol did not prevent tilt-induced vasodilatation, syncope or elevated plasma adrenaline.