Dysregulated Choline, Methionine, and Aromatic Amino Acid Metabolism in Patients with Wilson Disease: Exploratory Metabolomic Profiling and Implications for Hepatic and Neurologic Phenotypes.

Wilson disease (WD) is a genetic copper overload condition characterized by hepatic and neuropsychiatric symptoms with a not well-understood pathogenesis. Dysregulated methionine cycle is reported in animal models of WD, though not verified in humans. Choline is essential for lipid and methionine metabolism. Defects in neurotransmitters as acetylcholine, and biogenic amines are reported in WD; however, less is known about their circulating precursors. We aimed to study choline, methionine, aromatic amino acids, and phospholipids in serum of WD subjects. Hydrophilic interaction chromatography-quadrupole time-of-flight mass spectrometry was employed to profile serum of WD subjects categorized as hepatic, neurologic, and pre-clinical. Hepatic transcript levels of genes related to choline and methionine metabolism were verified in the Jackson Laboratory toxic milk mouse model of WD (tx-j). Compared to healthy subjects, choline, methionine, ornithine, proline, phenylalanine, tyrosine, and histidine were significantly elevated in WD, with marked alterations in phosphatidylcholines and reductions in sphingosine-1-phosphate, sphingomyelins, and acylcarnitines. In tx-j mice, choline, methionine, and phosphatidylcholine were similarly dysregulated. Elevated choline is a hallmark dysregulation in WD interconnected with alterations in methionine and phospholipid metabolism, which are relevant to hepatic steatosis. The elevated phenylalanine, tyrosine, and histidine carry implications for neurologic manifestations and are worth further investigation

Dysregulated Choline, Methionine, and Aromatic Amino Acid Metabolism in Patients with Wilson Disease: Exploratory Metabolomic Profiling and Implications for Hepatic and Neurologic Phenotypes

Wilson disease (WD) is a genetic copper overload condition characterized by hepatic and neuropsychiatric symptoms with a not well-understood pathogenesis. Dysregulated methionine cycle is reported in animal models of WD, though not verified in humans. Choline is essential for lipid and methionine metabolism. Defects in neurotransmitters as acetylcholine, and biogenic amines are reported in WD; however, less is known about their circulating precursors. We aimed to study choline, methionine, aromatic amino acids, and phospholipids in serum of WD subjects. Hydrophilic interaction chromatography-quadrupole time-of-flight mass spectrometry was employed to profile serum of WD subjects categorized as hepatic, neurologic, and pre-clinical. Hepatic transcript levels of genes related to choline and methionine metabolism were verified in the Jackson Laboratory toxic milk mouse model of WD (tx-j). Compared to healthy subjects, choline, methionine, ornithine, proline, phenylalanine, tyrosine, and histidine were significantly elevated in WD, with marked alterations in phosphatidylcholines and reductions in sphingosine-1-phosphate, sphingomyelins, and acylcarnitines. In tx-j mice, choline, methionine, and phosphatidylcholine were similarly dysregulated. Elevated choline is a hallmark dysregulation in WD interconnected with alterations in methionine and phospholipid metabolism, which are relevant to hepatic steatosis. The elevated phenylalanine, tyrosine, and histidine carry implications for neurologic manifestations and are worth further investigation

Metabolomics profiles of patients with Wilson disease reveal a distinct metabolic signature.

IntroductionWilson disease (WD) is characterized by excessive intracellular copper accumulation in liver and brain due to defective copper biliary excretion. With highly varied phenotypes and a lack of biomarkers for the different clinical manifestations, diagnosis and treatment can be difficult.ObjectiveThe aim of the present study was to analyze serum metabolomics profiles of patients with Wilson disease compared to healthy subjects, with the goal of identifying differentially abundant metabolites as potential biomarkers for this condition.MethodsHydrophilic interaction liquid chromatography-quadrupole time of flight mass spectrometry was used to evaluate the untargeted serum metabolome of 61 patients with WD (26 hepatic and 25 neurologic subtypes, 10 preclinical) compared to 15 healthy subjects. We conducted analysis of covariance with potential confounders (body mass index, age, sex) as covariates and partial least-squares analysis.ResultsAfter adjusting for clinical covariates and multiple testing, we identified 99 significantly different metabolites (FDR < 0.05) between WD and healthy subjects. Subtype comparisons also revealed significantly different metabolites compared to healthy subjects: WD hepatic subtype (67), WD neurologic subtype (57), WD hepatic-neurologic combined (77), and preclinical (36). Pathway analysis revealed these metabolites are involved in amino acid metabolism, the tricarboxylic acid cycle, choline metabolism, and oxidative stress.ConclusionsPatients with WD are characterized by a distinct metabolomics profile providing new insights into WD pathogenesis and identifying new potential diagnostic biomarkers

Wilson Disease: Intersecting DNA Methylation and Histone Acetylation Regulation of Gene Expression in a Mouse Model of Hepatic Copper Accumulation.

Background & aimsThe pathogenesis of Wilson disease (WD) involves hepatic and brain copper accumulation resulting from pathogenic variants affecting the ATP7B gene and downstream epigenetic and metabolic mechanisms. Prior methylome investigations in human WD liver and blood and in the Jackson Laboratory (Bar Harbor, ME) C3He-Atp7btx-j/J (tx-j) WD mouse model revealed an epigenetic signature of WD, including changes in histone deacetylase (HDAC) 5. We tested the hypothesis that histone acetylation is altered with respect to copper overload and aberrant DNA methylation in WD.MethodsWe investigated class IIa HDAC4 and HDAC5 and H3K9/H3K27 histone acetylation in tx-j mouse livers compared with C3HeB/FeJ (C3H) control in response to 3 treatments: 60% kcal fat diet, D-penicillamine (copper chelator), and choline (methyl group donor). Experiments with copper-loaded hepatoma G2 cells were conducted to validate in vivo studies.ResultsIn 9-week tx-j mice, HDAC5 levels increased significantly after 8 days of a 60% kcal fat diet compared with chow. In 24-week tx-j mice, HDAC4/5 levels were reduced 5- to 10-fold compared with C3H, likely through mechanisms involving HDAC phosphorylation. HDAC4/5 levels were affected by disease progression and accompanied by increased acetylation. D-penicillamine and choline partially restored HDAC4/5 and H3K9ac/H3K27ac to C3H levels. Integrated RNA and chromatin immunoprecipitation sequencing analyses revealed genes regulating energy metabolism and cellular stress/development, which, in turn, were regulated by histone acetylation in tx-j mice compared with C3H mice, with Pparα and Pparγ among the most relevant targets.ConclusionsThese results suggest dietary modulation of class IIa HDAC4/5, and subsequent H3K9/H3K27 acetylation/deacetylation can regulate gene expression in key metabolic pathways in the pathogenesis of WD

mtDNA depletion-like syndrome in Wilson disease.

Background and aimsWilson disease (WD) is caused by mutations in the copper transporter ATP7B, with its main pathology attributed to copper-mediated oxidative damage. The limited therapeutic effect of copper chelators and the early occurrence of mitochondrial deficits, however, undermine the prevalence of this mechanism.MethodsWe characterized mitochondrial DNA copy number and mutations as well as bioenergetic deficits in blood from patients with WD and in livers of tx-j mice, a mouse model of hepatic copper accumulation. In vitro experiments with hepatocytes treated with CuSO4 were conducted to validate in vivo studies.ResultsHere, for the first time, we characterized the bioenergetic deficits in WD as consistent with a mitochondrial DNA depletion-like syndrome. This is evidenced by enriched DNA synthesis/replication pathways in serum metabolomics and decreased mitochondrial DNA copy number in blood of WD patients as well as decreased mitochondrial DNA copy number, increased citrate synthase activity, and selective Complex IV deficit in livers of the tx-j mouse model of WD. Tx-j mice treated with the copper chelator penicillamine, methyl donor choline or both ameliorated mitochondrial DNA damage but further decreased mitochondrial DNA copy number. Experiments with copper-loaded HepG2 cells validated the concept of a direct copper-mitochondrial DNA interaction.ConclusionsThis study underlines the relevance of targeting the copper-mitochondrial DNA pool in the treatment of WD separate from the established copper-induced oxidative stress-mediated damage

mtDNA depletion-like syndrome in Wilson disease.

Background and aimsWilson disease (WD) is caused by mutations in the copper transporter ATP7B, with its main pathology attributed to copper-mediated oxidative damage. The limited therapeutic effect of copper chelators and the early occurrence of mitochondrial deficits, however, undermine the prevalence of this mechanism.MethodsWe characterized mitochondrial DNA copy number and mutations as well as bioenergetic deficits in blood from patients with WD and in livers of tx-j mice, a mouse model of hepatic copper accumulation. In vitro experiments with hepatocytes treated with CuSO4 were conducted to validate in vivo studies.ResultsHere, for the first time, we characterized the bioenergetic deficits in WD as consistent with a mitochondrial DNA depletion-like syndrome. This is evidenced by enriched DNA synthesis/replication pathways in serum metabolomics and decreased mitochondrial DNA copy number in blood of WD patients as well as decreased mitochondrial DNA copy number, increased citrate synthase activity, and selective Complex IV deficit in livers of the tx-j mouse model of WD. Tx-j mice treated with the copper chelator penicillamine, methyl donor choline or both ameliorated mitochondrial DNA damage but further decreased mitochondrial DNA copy number. Experiments with copper-loaded HepG2 cells validated the concept of a direct copper-mitochondrial DNA interaction.ConclusionsThis study underlines the relevance of targeting the copper-mitochondrial DNA pool in the treatment of WD separate from the established copper-induced oxidative stress-mediated damage