Ronald J. A. Wanders

ACBD5 deficiency causes a defect in peroxisomal very long-chain fatty acid metabolism

Background Acyl-CoA binding domain containing protein 5 (ACBD5) is a peroxisomal membrane protein with a cytosolic acyl-CoA binding domain. Because of its acyl-CoA binding domain, ACBD5 has been assumed to function as an intracellular carrier of acyl-CoA esters. In addition, a role for ACBD5 in pexophagy has been suggested. However, the precise role of ACBD5 in peroxisomal metabolism and/or functioning has not yet been established. Previously, a genetic ACBD5 deficiency was identified in three siblings with retinal dystrophy and white matter disease. We identified a pathogenic mutation in ACBD5 in another patient and studied the consequences of the ACBD5 defect in patient material and in ACBD5-deficient HeLa cells to uncover this role. Methods We studied a girl who presented with progressive leukodystrophy, syndromic cleft palate, ataxia and retinal dystrophy. We performed biochemical, cell biological and molecular studies in patient material and in ACBD5-deficient HeLa cells generated by CRISPR-Cas9 genome editing. Results We identified a homozygous deleterious indel mutation in ACBD5, leading to complete loss of ACBD5 protein in the patient. Our studies showed that ACBD5 deficiency leads to accumulation of very long-chain fatty acids (VLCFAs) due to impaired peroxisomal β-oxidation. No effect on pexophagy was found. Conclusions Our investigations strongly suggest that ACBD5 plays an important role in sequestering C26-CoA in the cytosol and thereby facilitates transport into the peroxisome and subsequent β-oxidation. Accordingly, ACBD5 deficiency is a novel single peroxisomal enzyme deficiency caused by impaired VLCFA metabolism, leading to retinal dystrophy and white matter disease.

Zellweger syndrome with unusual findings: non-immune hydrops fetalis, dermal erythropoiesis and hypoplastic toe nails

The peroxisomal biogenesis disorders (PBDs) comprise the Zellweger spectrum disorders (i.e., Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease) and rhizomelic chondrodysplasia punctata. Peroxisomal biogenesis disorders can be caused by mutations in any of 13 currently known PEX genes, which encode peroxins involved in peroxisomal protein import and/or assembly of the organelle. We report here on a Turkish patient who presented with unusual clinical findings, that included non-immune hydrops, dermal erythropoiesis and hypoplastic toenails, as well as common dysmorphic features of Zellweger syndrome. The patient has also pulmonary hypoplasia, which has been reported in only a few patients with Zellweger syndrome. A peroxisomal biogenesis disorder was confirmed by enzyme analysis and abnormal very long-chain fatty acid (VLCFA) profiles in plasma and fibroblast and immunofluorescence microscopy studies. Subsequent molecular genetic analysis revealed a homozygous c.856C>T mutation (R268X) in the PEX3 gene, which made this patient the third to have a defect in this gene. In contrast to those of the two previously reported patients, the cells of this patient still contained peroxisomal membrane structures (ghosts), seen by immunofluorescence microscopy analysis. The case presented here and the two previously reported cases point out that a PEX3 gene defect may present with fairly heterogeneous clinical findings. This case also raises a possibility that hydrops fetalis may be associated with a PEX3 gene defect and that peroxisomal disorders can be considered in the etiology of hydrops fetalis as well as other cell organelle disorders when one is considering yet undiscovered complementation groups in peroxisomal disorders.

Inhibition of 3-methylcrotonyl-CoA carboxylase explains the increased excretion of 3-hydroxyisovaleric acid in valproate-treated patients

BackgroundValproic acid (VPA) is a widely used anticonvulsant drug which affects mitochondrial metabolism including the catabolism of fatty acids and branched-chain amino acids.AimsTo elucidate the effect of valproate on the leucine pathway through a targeted metabolomics approach and the evaluation of the effects of valproate on the activity of biotinidase and 3-methylcrotonyl-CoA carboxylase (3MCC).MethodsUrine organic acid analysis was performed in patients under VPA therapy and healthy controls using gas-chromatography/mass spectrometry (GC-MS). Biotinidase activity was determined in plasma samples of both groups using an optimized spectrophotometric assay. After immunoprecipitation of short-chain enoyl-CoA hydratase (crotonase, ECHS1), 3MCC activity was measured in human liver homogenate using high-performance liquid chromatography (HPLC), in the absence and presence of valproyl-CoA.ResultsThe levels of 3-hydroxyisovaleric acid (3OH-IVA), one secondary metabolite of the leucine pathway, were significantly elevated in human urine after VPA treatment. Biotinidase activity in plasma samples ranged from very low to normal levels in treated patients as compared with controls. Enzyme activity measurements revealed inhibition of 3-methylcrotonyl-CoA carboxylase by valproyl-CoA (IC50u2009=u20091.36xa0mM). Furthermore, we show that after complete immunoprecipitation of crotonase in a human liver homogenate, 3-hydroxyisovaleryl-CoA is not formed.DiscussionOur results suggest the interference of VPA with the activity of 3MCC through a potential cumulative effect: direct inhibition of the enzyme activity by the drug metabolite valproyl-CoA and the inhibition of biotinidase by valproate and/or its metabolites. These interactions may be associated with the skin rash and hair loss which are side effects often reported in VPA-treated patients.

Ketones and inborn errors of metabolism: old friends revisited

During exercise fat and carbohydrates are the principal substrates that fuel aerobic ATP synthesis in human skeletal muscle. During low-intensity exercise, skeletal muscle relies predominantly on fat-based fuels, thereby sparing the intracellular glycogen stores. During high-intensity exercise, however, there is a shift in fuel selection from fat towards glycolysis and the utilization of glycogen reserves. When glycogen reserves turn low during prolonged intense exercise, but also upon fasting and starvation, ketone bodies such as 3-βhydroxybutyrate (β-HB) and acetoacetate which are produced by the liver, are increasingly important sources of ATP (Fig. 1). A paper by Cox and colleagues in Cell Metabolism (Cox et al 2016) highlights the benefit of ketone bodies in healthy subjects. Well-trained athletes were supplemented with a synthesized ketone ester ((R)-3-hydroxybutyl (R)-3hydroxybutyrate ketone). This ketone ester led to the reprogramming of skeletal muscle metabolic pathways during exercise and acutely improved cycling performance in these athletes. The ingested ketone ester is converted intoβ-HB and (R)-1,3-butanediol by nonspecific gut esterases, after which the liver converts (R)-1,3-butanediol into another molecule of β-HB. Skeletal muscle mono-carboxylate transporters subsequently transport β-HB across the plasma membrane and into the mitochondria. The increased ketone body levels preserve intramuscular glycogen stores and branched-chain amino acids (BCAA) and reduce lactate release during exercise (Fig. 1; Cox et al 2016). The ketone ester developed by Cox and coworkers may be of great benefit for patients with certain inborn errors of metabolism (IEMs) by serving as an alternative energy source for ATP production (Fig. 1). In addition, patients in which ketone body formation is impaired, such as HMG-CoA synthase deficiency, HMG-CoA lyase deficiency and beta-ketothiolase deficiency, could benefit from this ketone ester. A special IEM that would qualify for ketone ester treatment would be SUCLA deficiency in which the formation of succinate from succinyl-CoA in the TCA cycle is impaired. An increased flux through succinyl-CoA acetoacetate transferase by ketone ester treatment would, at least in theory, increase flux through the TCA cycle. The idea of treating patients with ketone bodies or ketogenic diets is not new (reviewed in (Scholl-Burgi et al 2015)). In glucose transporter type 1 deficiency (GLUT1) and pyruvate dehydrogenase complex (PDHc) deficiency, ketogenic diets are part of the therapy of choice (Scholl-Burgi et al 2015). Some mitochondrial complex I and II deficiencies could benefit from this ketone ester (Scholl-Burgi et al 2015). A ketogenic diet in patients with glycogen storage disease type III and V significantly improved cardiac remodelling (Valayannopoulos et al 2011) and exercise tolerance (Vorgerd and Zange 2007). Long-term D,L-3-hydroxybutyrate treatment improved cardiac contractility in infants with multiple acylCoA dehydrogenase deficiency (Van Hove et al 2003). The newly synthesized ketone ester is preferred over βHB itself, however, since supplementation of the latter is associated with a substantial load of either acid and/or salt. Also, a ketone drink is easier to adhere to than a ketogenic (high fat, low carbohydrate) diet. As such, this ketone ester could serve as an attractive therapeutic option in * Riekelt H. Houtkooper r.h.houtkooper@amc.uva.nl

CHAPTER 14:Phytanoyl-CoA Hydroxylase: A 2-Oxoglutarate-Dependent Dioxygenase Crucial for Fatty Acid Alpha-Oxidation in Humans

Phytanoyl-CoA hydroxylase belongs to the family of 2-oxoglutarate-dependent dioxygenases and plays a crucial role in the α-oxidation of fatty acids. The complete α-oxidation pathway involves five different enzymes localized in peroxisomes. Thus far, phytanoyl-CoA hydroxylase deficiency has remained the only genetically determined inborn error of metabolism affecting the α-oxidation pathway. In this chapter we describe the current state of knowledge on fatty acid α-oxidation with special emphasis on phytanoyl-CoA hydroxylase and its deficiency in Refsum disease.

Essential Fatty Acid Deficiency In Very Long-Chain Acyl-Coa Dehydrogenase Deficient Patients

Introduction: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), a long-chain fatty acid (LCFA) beta-oxidation dis order, may be treated with LCFA restriction. As Essential Fatty Acids (EFAs) are LCFAs, patients may be at risk for EFA deficiency. Objectives: Investigate whether LCFA restrictions lead to EFA deficiency in VLCADD and which markers are indicative of EFA deficiency. Methods: Thirty-nine LCFA profiles of 16 VLCADD patients were determined in erythrocytes and compared to 48 healthy controls. The predictive value of EFA deficiency markers was calculated from data of a historic cohort (n=4523, 0-39yrs). Results: Linoleic acid (LA), dihomo-γ-linolenic acid (DHLA) and eicosapentaenoic acid (EPA) were significantly decreased in VLCADD patients. Patients on docosahexaenoic acid (DHA) and arachidonic acid (AA) supplementation exhibited even lower LA. Mead acid, a presumed marker for EFA-deficiency, was not increased in patients. In the historic cohort, sensitivity of MA was low for LA deficiency (24% for levels <2.5 percentile) and for DHA+AA deficiency (12% for levels <2.5 percentile). Discussion: VLCADD patients on LCFA restriction are prone to develop LA deficiency. Furthermore, MA is a specific, but not a sensitive marker for LA or EFA deficiency, neither in VLCADD patients, nor in healthy controls, nor in a large patient cohort.

Very-Long-Chain Fatty Acids and Phytanic Acid

The peroxisomes are subcellular organelles with a variety of biochemical functions, amongst which a system for beta-oxidation of very long-chain (C22-C26) fatty acids (VLCFA) and a system for alpha-oxidation of branched-chain fatty acids such as phytanic acid. The plasma concentrations of the VLCFA as well as those of phytanic acid and its immediate metabolite pristanic acid are important biomarkers for the assessment of peroxisomal dysfunction. A gas chromatography / mass spectrometry analysis of the tertiarybutyl-dimethylsilyl esters of the afore mentioned acids, using stable isotope labeled internal standards, is the method of choice for the diagnosis of peroxisome biogenesis defects (Zellweger spectrum patients) as well as isolated peroxisomal enzyme defects such as X-linked adrenoleucodystrophy, D-bifunctional protein deficiency and Refsum disease, amongst others. This approach is equally well suited for therapy monitoring of treatable disorders.