Straight-chain acyl-CoA Oxidase Research Articles

A de novo heterozygous variant in ACOX1 gene cause Mitchell syndrome: the first case in China and literature review

BackgroundMitchell syndrome (MITCH) is a rare autosomal dominant hereditary disorder, characterized by episodic demyelination, sensorimotor polyneuropathy and hearing loss. MITCH is caused by heterozygous mutation in the ACOX1 gene, which encodes straight-chain acyl-CoA oxidase, on chromosome 17q25.1. Only 5 unrelated patients have been reported so far, and no reports from China. Here, we describe the first MITCH case in a Chinese individual.Case presentationA 7-year-old girl initially presented with diffuse desquamatory rash at age 3. Her clinical symptoms in order of presentation were diffuse desquamatory rash, gait instability, ptosis with photophobia, hearing loss, abdominal pain, diarrhea, nausea, and dysuria. Genetic analysis demonstrated that the patient carried a heterozygous variant c.710A>G(p.Asp237Ser) in the ACOX1 gene, which can cause MITCH symptoms. This is the first MITCH case with gastrointestinal and urinary tract symptoms. After administrating N acetylcysteine amide (NACA), some symptoms were relieved and the patient’s condition improved.ConclusionThis is the first MITCH case in the Chinese population, and we expanded the genotype spectrum of it. The p.Asp237Ser may be a mutational hotspot in ACOX1 regardless of race. In terms of diagnosis, patients with recurrent rash, gait instability, and hearing loss with some autonomic symptoms should raise the suspicion of MITCH and proper and prompt treatment should be given.

The biosynthesis of DHA is increased in the liver of diabetic rats induced by high‐fat diets and STZ, in correlation with increased activity of peroxisomal β‐oxidation

Docosahexaenoic acid (DHA) biosynthesis in the liver is the most important source of extra‐hepatic tissue DHA. However, the levels of DHA and DHA biosynthesis in the livers of animals with type 2 diabetes mellitus remain unknown. In the current study, we used type 2 diabetic rats induced by high‐fat diets and low‐dose streptozotocin (STZ, 25 mg/kg) to investigate DHA biosynthesis in the liver during type 2 diabetes mellitus. The serum and livers were collected. Biochemical characteristics, fatty acid profiles, the activity of peroxisomal β‐oxidation, and the expressions of enzymes involved in the DHA biosynthesis pathway were assayed in the liver of diabetic rats. The data showed that DHA levels were unexpectedly increased despite of the lower expressions of key enzymes, Δ5‐ and Δ6‐desaturases (Δ5D and Δ6D) in the endoplasmic reticulum (ER). The increased DHA was accompanied by an increase in the expression of straight‐chain acyl‐CoA oxidase (SCOX), D‐bifunctional protein (DBP), and 3‐oxoacyl‐CoA thiolase (THL), as well as elevated activity of peroxisomal β‐oxidation and an increased capacity for DHA biosynthesis from C24:6n‐3. These findings indicate that DHA biosynthesis is increased in the livers of diabetic rats induced by high‐fat diets and STZ, and that this effect is related to increased activity of peroxisomal β‐oxidation, but not of ER desaturases.Practical applications: The results support the hypothesis that the dietary supplementation in diabetes with DHA and EPA is desirable.The pathway of DHA biosynthesis in the liver of rats induced by high‐fat diets and STZ is shown. DHA biosynthesis is increased in the livers of diabetic rats induced by high‐fat diets and STZ, and this effect is related to increased activity of peroxisomal β‐oxidation, but is not related to the activity of ER desaturases.

Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein

Compared with other mammalian tissues, retina is highly enriched in PUFA. Long-chain PUFA (LC-PUFA; C18-C24) are essential FAs that are enriched in the retina and are necessary for maintenance of normal retinal development and function. The retina, brain, and sperm also contain very LC-PUFA (VLC-PUFA; >C24). Although VLC-PUFA were discovered more than two decades ago, very little is known about their biosynthesis and functional roles in the retina. This is due mainly to intrinsic difficulties associated with working on these unusually long polyunsaturated hydrocarbon chains and their existence in small amounts. Recent studies on the FA elongase elongation of very long chain fatty acids-4 (ELOVL4) protein, however, suggest that VLC-PUFA probably play some uniquely important roles in the retina as well as the other tissues. Mutations in the ELOVL4 gene are found in patients with autosomal dominant Stargardt disease. Here, we review the recent literature on VLC-PUFA with special emphasis on the elongases responsible for their synthesis. We focus on a novel elongase, ELOVL4, involved in the synthesis of VLC-PUFA, and the importance of these FAs in maintaining the structural and functional integrity of retinal photoreceptors.

Bile acids: the role of peroxisomes

It is well established that peroxisomes play a crucial role in de novo bile acid synthesis. Studies in patients with a peroxisomal disorder have been indispensable for the elucidation of the precise role of peroxisomes. Several peroxisomal disorders are associated with distinct bile acid abnormalities and each disorder has a characteristic pattern of abnormal bile acids that accumulate, which is often used for diagnostic purposes. The patients have also been important for determining the pathophysiological consequences of defects in bile acid biosynthesis. In this review, we will discuss all the peroxisomal steps involved in bile acid synthesis and the bile acid abnormalities in patients with peroxisomal disorders. We will show the results of bile acid measurements in several tissues from patients, including brain, and we will discuss the toxicity and the pathological effects of the abnormal bile acids.

Peroxisomal acyl‐CoA‐oxidase deficiency: Two new cases

We report on two new patients with straight-chain acyl-coenzyme A oxidase deficiency. Early onset hypotonia, seizures and psychomotor delay were observed in both cases. Plasma very-long-chain fatty acids were abnormal in both patients, whereas the plasma levels of phytanic acid, pristanic acid, the bile acid intermediates DHCA and THCA, and erythrocyte plasmalogen levels were normal. Studies in fibroblasts from the two patients revealed a deficiency of one of the two peroxisomal acyl-CoA oxidases, that is, straight-chain acyl-CoA oxidase (ACOX1). Subsequent molecular analysis of ACOX1 showed a homozygous deletion, which removes a large part of intron 3 and exons 4-14 in the first patient. Mutation analysis in the second patient revealed compound heterozygosity for two mutations, including: (1) a c.692 G > T (p.G231V) mutation and (2) skipping of exon 13 (c.1729_1935del (p.G577_E645del).

Clinical, biochemical, and mutational spectrum of peroxisomal acyl–coenzyme A oxidase deficiency

Peroxisomal acyl-coenzyme A (acyl-CoA) oxidase deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation due to a deficiency of straight-chain acyl-CoA oxidase (SCOX). The biochemical hallmark of this disorder is the accumulation of very long-chain fatty acids. Although some case reports and small series of patients have been published, a comprehensive overview of the clinical, biochemical, and mutational spectrum of this disorder is still lacking. For this reason, we report clinical information for a cohort of 22 patients with peroxisomal acyl-CoA oxidase deficiency and the results from biochemical and mutation analyses in fibroblasts of the patients. No clear genotype-phenotype correlation was observed. An intriguing mutation in the alternatively-spliced transcript encoding the isoform SCOX-exon 3II in a patient with normal expression of the transcript encoding the isoform SCOX-exon 3I, prompted us to characterize these two isoforms of human SCOX. The recombinant SCOX-exon 3I displayed activity toward medium-chain fatty acyl-CoAs and was not active with very long-chain fatty acyl-CoAs. In contrast, recombinant SCOX-exon 3II was capable of oxidizing a broad range of substrates, including very long-chain fatty acyl-CoAs. These results explain why this patient with a mutation in exon 3II of the ACOX1 gene, but with normal expression of exon 3I, was indistinguishable from other patients with peroxisomal acyl-CoA oxidase deficiency with respect to his clinical presentation and the biochemical abnormalities in his fibroblasts.

Mutations in the Gene Encoding Peroxisomal Sterol Carrier Protein X (SCPx) Cause Leukencephalopathy with Dystonia and Motor Neuropathy

In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7).

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARα-dependent and -independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.

Identification of the peroxisomal β-oxidation enzymes involved in the degradation of long-chain dicarboxylic acids

Dicarboxylic acids (DCAs) are omega-oxidation products of monocarboxylic acids. After activation by a dicarboxylyl-CoA synthetase, the dicarboxylyl-CoA esters are shortened via beta-oxidation. Although it has been studied extensively where this beta-oxidation process takes place, the intracellular site of DCA oxidation has remained controversial. Making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this paper that peroxisomes, and not mitochondria, are involved in the beta-oxidation of C16DCA. Additional studies in fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, together with direct enzyme measurements with human recombinant l-bifunctional protein (LBP) and DBP expressed in a fox2 deletion mutant of Saccharomyces cerevisiae, show that the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP, and sterol carrier protein X, possibly together with the classic 3-ketoacyl-CoA thiolase. This is the first indication of a specific function for LBP, which has remained elusive until now.

Evidence for increased oxidative stress in peroxisomal D-bifunctional protein deficiency

Peroxisome biogenesis disorders (PBDs) and D-bifunctional protein (D-BP) deficiency are two types of inherited peroxisomal disorders. Patients with a PBD lack functional peroxisomes and patients with D-BP deficiency lack the enzyme, which is responsible for the second and third step of the peroxisomal β-oxidation. The clinical presentation of these peroxisomal disorders is severe and includes several neurological abnormalities. The pathological mechanisms underlying these disorders are not understood and no therapies are available. Because peroxisomes have been associated with oxidative stress, as oxygen radicals are both produced and scavenged in peroxisomes, we have investigated whether oxidative stress is involved in the pathogenesis of PBDs and D-BP deficiency. We found in D-BP-deficient patients increased levels of thiobarbituric acid-reactive substances (TBARS) and 8-hydroxydeoxyguanosine (8-OHdG), which are markers for lipid peroxidation and oxidative DNA damage, respectively, whereas the levels of the lipophilic antioxidants α-tocopherol and coenzyme Q 10 were decreased. In addition, we found in skin fibroblasts from D-BP-deficient patients an imbalance between the activities of the peroxisomal H 2O 2-generating straight-chain acyl-CoA oxidase (SCOX) and the peroxisomal H 2O 2-degrading enzyme catalase. In conclusion, we have found clear evidence for the presence of increased oxidative stress in patients with D-BP deficiency, but not in patients with a PBD.

Reinvestigation of Peroxisomal 3-Ketoacyl-CoA Thiolase Deficiency: Identification of the True Defect at the Level of d-Bifunctional Protein

In this report, we reinvestigate the only patient ever reported with a deficiency of peroxisomal 3-ketoacyl-CoA thiolase (THIO). At the time when they were described, the abnormalities in this patient, which included accumulation of very-long-chain fatty acids and the bile-acid intermediate trihydroxycholestanoic acid, were believed to be the logical consequence of a deficiency of the peroxisomal beta-oxidation enzyme THIO. In light of the current knowledge of the peroxisomal beta-oxidation system, however, the reported biochemical aberrations can no longer be explained by a deficiency of this thiolase. In this study, we show that the true defect in this patient is at the level of d-bifunctional protein (DBP). Immunoblot analysis revealed the absence of DBP in postmortem brain of the patient, whereas THIO was normally present. In addition, we found that the patient had a homozygous deletion of part of exon 3 and intron 3 of the DBP gene, resulting in skipping of exon 3 at the cDNA level. Our findings imply that the group of single-peroxisomal beta-oxidation-enzyme deficiencies is limited to straight-chain acyl-CoA oxidase, DBP, and alpha-methylacyl-CoA racemase deficiency and that there is no longer evidence for the existence of THIO deficiency as a distinct clinical entity.

Straight-Chain Acyl-CoA Oxidase Knockout Mouse Accumulates Extremely Long Chain Fatty Acids from α-Linolenic Acid: Evidence for Runaway Carousel-Type Enzyme Kinetics in Peroxisomal β-Oxidation Diseases

Extremely long chain polyunsaturated fatty acids (ELCPs) with >24 carbons and four or more double bonds are normally found in excitatory tissues but have no known function, and are greatly increased in brain and other tissues of humans with peroxisomal disorders. Straight-chain acyl-CoA oxidase (AOX) catalyzes the first, rate-limiting step of peroxisomal β-oxidation of very-long-chain saturated and unsaturated fatty acids. We have studied the polyunsaturated fatty acid metabolism of AOX knockout mice (AOX−/−) as a model of human AOX deficiency (pseudo-neonatal adrenoleukodystrophy), and as a genetic tool to test the putative peroxisomal β-oxidation involvement in polyunsaturated fatty acid synthesis. Liver lipids of 26-day-old weanling AOX−/− mice livers accumulate n-3 and n-6 ELCPs from C24 to C30 with 5 and 6 double bonds, have 356 ± 66 μg/g docosahexaenoic acid (22:6n-3), similar to congenic (AOX−/* = AOX+/+ and AOX+/−) controls (401 ± 96 μg/g), but increased 22:5n-6 (22.4 ± 3.7 vs 6.4 ± 1.5 μg/g). AOX+/* mice injected intraperitoneally at 23 days with [U-13C]-18:3n-3show strong labeling of 22:6n-3 after 72 h, whereas AOX−/− mice display less labeling of 22:6n-3but strong tracer incorporation into 24:6n-3, 26:6n-3, and 28:6n-3, after the same period. These data suggest that ELCPs are natural runaway elongation by-products of 22:6n-3 and 22:5n-6 synthesis, which are normally disposed of by peroxisomal β-oxidation. Under conditions with impaired peroxisomal β-oxidation, such as Zellweger syndrome and adrenoleukodystrophies, ELCPs accumulate due to increased synthesis and impaired disposal. Two mechanisms for the formation of these runaway elongation by-products and the involvement of secondary carnitine deficiency in this process are proposed: n-3 ELCPs are synthesized by a carnitine-dependent multifunctional mitochondrial docosahexaenoic acid synthase (mtDHAS) which normally synthesizes primarily 22:6n-3, while n-6 ELCPs are synthesized by independent elongation enzymes in the endoplasmic reticulum.

Identification of the peroxisomal β-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid

DHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by beta-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the beta-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in beta-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal beta-oxidation.

Immunocytochemical localization of acyl-CoA oxidase in the rat central nervous system.

Peroxisomal beta-oxidation, consisting of four steps catalysed by an acyl-CoA oxidase, a multifunctional protein and a thiolase, is responsible for the shortening of a variety of lipid compounds. The first reaction of this pathway is catalysed by a FAD-containing acyl-CoA oxidase, three isotypes of which have been so far recognised. Among these, straight-chain acyl-CoA oxidase (ACOX) acts on long and very long chain fatty acids, prostaglandins and some xenobiotics. We investigated ACOX localisation by means of a sensitive, tyramide based, immunocytochemical technique, thus obtaining a complete distribution atlas of the enzyme in adult rat CNS. Granular immunoreaction product was found in the cytoplasm of neuronal and glial cells, both in the perikarya and in the cell processes. ACOX immunoreactive neurons were present to variable extent, in either forebrain or hindbrain areas. Specifically, the strongest signal was detected in the pallidum, septum, red nucleus, reticular formation, nuclei of the cranial nerves, and motoneurons of the spinal cord. We then compared the ACOX immunoreactivity pattern with our previous distribution maps of other peroxisomal enzymes in the adult rat brain. While ACOX appeared to colocalise with catalase in the majority of cerebral regions, some differences with respect to d-amino acid oxidase were noted. These observations support the hypothesis of heterogeneous peroxisomal populations in the nervous tissue. The wide distribution of the enzyme in the brain is consistent with the severe and generalised neurological alterations characterising the peroxisomal disorder caused by ACOX deficiency (pseudo-neonatal adrenoleukodystrophy).

CDNA cloning and analysis of tissue-specific expression of mouse peroxisomal straight-chain acyl-CoA oxidase.

Straight-chain acyl-CoA oxidase is the first and rate limiting enzyme in the peroxisomal beta-oxidation pathway catalysing the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, thereby producing H2O2. To study peroxisomal beta-oxidation we cloned and characterized the cDNA of mouse peroxisomal acyl-CoA oxidase. It consists of 3778 bp, including a 1983-bp ORF encoding a polypeptide of 661 amino-acid residues. Like the rat and human homologue the C-terminus contains an SKL motif, an import signal present in several peroxisomal matrix proteins. Sequence analysis revealed high amino-acid homology with rat (96%) and human (87%) acyl-CoA oxidase in addition to minor homology ( approximately 40%) with other related proteins, such as rabbit trihydroxy-cholestanoyl-CoA oxidase, human branched chain acyl-CoA oxidase and rat trihydroxycoprostanoyl-CoA oxidase. Acyl-CoA oxidase mRNA and protein expression were most abundant in liver followed by kidney, brain and adipose tissue. During mouse brain development acyl-CoA oxidase mRNA expression was highest during the suckling period indicating that peroxisomal beta-oxidation is most critical during this developmental stage. Comparing tissue mRNA levels of peroxisome proliferator-activated receptor alpha and acyl-CoA oxidase, we noticed a constant relationship in all tissues investigated, except heart and adipose tissue in which much more, and respectively, much less, peroxisome proliferator-activated receptor alpha mRNA in proportion to acyl-CoA oxidase mRNA was found. Our data show that acyl-CoA oxidase is an evolutionary highly conserved enzyme with a distinct pattern of expression and indicate an important role in lipid metabolism.

Complementation Analysis of Fibroblasts from Peroxisomal Fatty Acid Oxidation Deficient Patients Shows High Frequency of Bifunctional Enzyme Deficiency Plus Intragenic Complementation: Unequivocal Evidence for Differential Defects in the Same Enzyme Protein

In the last few years many patients have been reported with a defect in peroxisomal fatty acid β-oxidation of unknown origin. Using a combined approach based on direct activity measurements of straight-chain acyl-CoA oxidase and complementation analysis after somatic cell fusion of fibroblasts, we have now classified 13 patients into 4 distinct groups representing different gene defects. Remarkably, we found intragenic complementation in group 2 so that group 2 is in fact made up of 3 distinct subgroups. The underlying basis for this peculiar phenomenon probably has to do with the fact that bifunctional protein harborstwocatalytic activities including enoyl-CoA hydrataseand3-hydroxyacyl-CoA dehydrogenase. In group 2A enoyl-CoA hydrataseand3-hydroxyacyl-CoA dehydrogenase are defective whereas in group 2B and 2C either the hydrataseor3-hydroxyacyl-CoA dehydrogenase component of the bifunctional protein is deficient.