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Objective: Bile acid intermediates, 3α,7α,12α-trihydroxycholestanoic acid (THCA) and 3α,7α-dihydroxycholestanoic acid (DHCA), are metabolized in peroxisomes. Some peroxisomal disorders (PDs), such as Zellweger spectrum disorder (ZSD), show an accumulation of bile acid intermediates. In particular, ABCD3 deficiency and acyl-CoA-oxidase 2 deficiency are characterized by these metabolite abnormalities. In patients with ZSD, levels of bile acid intermediates can be lowered by a primary bile acid supplementation treatment; therefore, measuring their levels could help evaluate treatment effectiveness. Here, we established a method for the quantitative determination of bile acid intermediates (THCA/DHCA) for differentiating PDs and assessing bile acid treatment., Methods: Serum samples, obtained from patients with several forms of ZSD as well as peroxisomal β-oxidation enzyme deficiencies, were deproteinized and analyzed using liquid chromatography-mass spectrometry., Results: Levels of the bile acid intermediates increased significantly in patients with Zellweger syndrome (ZS) and slightly in patients with neonatal adrenoleukodystrophy and infantile Refsum disease (IRD), reflecting the severity of these diseases. One patient with ZS treated with primary bile acids for 6 months showed slightly decreased serum DHCA levels but significantly increased serum THCA levels. One patient with IRD who underwent living-donor liver transplantation showed a rapid decrease in serum THCA and DHCA levels, which remained undetected for 6 years. In all controls, THCA and DHCA levels were below the detection limit., Conclusion: The analytical method developed in this study is useful for diagnosing various PD and validating bile acid treatment. Additionally, it can help predict the prognosis of patients with PD and support treatment strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options., Competing Interests: Declaration of Competing Interest The authors declare no competing or financial interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In Zellweger syndrome (ZS), lack of peroxisome function causes physiological and developmental abnormalities in many organs such as the brain, liver, muscles, and kidneys, but little is known about the exact pathogenic mechanism. By disrupting the zebrafish pex2 gene, we established a disease model for ZS and found that it exhibits pathological features and metabolic changes similar to those observed in human patients. By comprehensive analysis of the fatty acid profile, we found organ-specific accumulation and reduction of distinct fatty acid species, such as an accumulation of ultra-very-long-chain polyunsaturated fatty acids (ultra-VLC-PUFAs) in the brains of pex2 mutant fish. Transcriptome analysis using microarray also revealed mutant-specific gene expression changes that might lead to the symptoms, including reduction of crystallin, troponin, parvalbumin, and fatty acid metabolic genes. Our data indicated that the loss of peroxisomes results in widespread metabolic and gene expression changes beyond the causative peroxisomal function. These results suggest the genetic and metabolic basis of the pathology of this devastating human disease., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest associated with this study., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Metabolic changes occur in patients with peroxisomal diseases owing to impairments in the genes involved in peroxisome function. For diagnostic purposes, saturated very-long-chain fatty acids (VLCFAs) such as C24:0 and C26:0, phytanic acid, pristanic acid, and plasmalogens are often measured as metabolic hallmarks. As the direct pathology of peroxisomal disease is yet to be fully elucidated, we sought to explore the fatty acid species that accumulate in patients with peroxisomal diseases. We developed a method for detecting a range of fatty acids implicated in peroxisomal diseases such as Zellweger syndrome (ZS) and X-linked adrenoleukodystrophy (X-ALD). To this end, we employed an ultra-performance liquid chromatography-mass spectrometry (LC-MS) coupled with negatively charged electrospray ionization. Fatty acids from patients and control subjects were extracted from total lipids by acid-hydrolysis and compared. In accordance with previous results, the amounts of VLCFAs, phytanic acid, and pristanic acid differed between the two groups. We identified extremely long and highly polyunsaturated VLCFAs (ultra-VLC-PUFAs) such as C44:12 in ZS samples. Moreover, three unknown molecules were prominent in control samples but scarcely detectable in ZS samples. LC-MS/MS analysis identified these as 1-alkyl-sn-glycerol 3-phosphates derived from ether lipids containing fatty alcohols such as C16:0, C18:0, or C18:1. Our method provides an approach to observing a wide range of lipid-derived fatty acids and related molecules in order to understand the metabolic changes involved in peroxisomal diseases. This technique can therefore be used in identifying metabolic markers and potential clinical targets for future treatment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Zellweger syndrome, one of the peroxisome biogenesis disorders, is an autosomal recessive disease caused by mutations in PEX genes. It is characterized by severe hypotonia, failure to thrive, psychomotor retardation, liver dysfunction, and sensorineural hearing impairment. Most of the patients with this disease die before the age of 1 year. PEX14 is the 13th PEX gene responsible for peroxisome biogenesis disorders. Thus far, only two patients with PEX14 deficiency have been reported. Here, we report the first case of a Japanese patient with a PEX14 mutation who showed severe hypotonia, psychomotor retardation, demyelination, and developed rickets at the age of 5 months. An increased excretion of 3,6-epoxydicarboxylic acids leads to the diagnosis of Zellweger syndrome and a mutation analysis of PEX14 revealed a homozygous mutation of c.538C>T (p.Q180X). The patient survived for a prolonged period of time but died of liver failure at the age of 46 months., (© 2015 Japan Pediatric Society.)

The ratio of C 26:0/C 22:0 fatty acids in patient lipids is widely accepted as a critical clinical criterion of peroxisomal diseases, such as Zellweger syndrome and X-linked adrenoleukodystrophy (X-ALD). However, phospholipid molecular species with very long chain fatty acids (VLCFA) have not been precisely characterized. In the present study, the structures of such molecules in fibroblasts of Zellweger syndrome and X-ALD were examined using LC-ESI-MS/MS analysis. In fibroblasts from Zellweger patients, a large number of VLCFA-containing molecular species were detected in several phospholipid classes as well as neutral lipids, including triacylglycerol and cholesteryl esters. Among these lipids, phosphatidylcholine showed the most diversity in the structures of VLCFA-containing molecular species. Some VLCFA possessed longer carbon chains and/or larger number of double bonds than C 26:0-fatty acid (FA). Similar VLCFA were also found in other phospholipid classes, such as phosphatidylethanolamine and phosphatidylserine. In addition, VLCFA-containing phospholipid species showed some differences among fibroblasts from Zellweger patients. It appears that phospholipids with VLCFA, with or without double bonds, as well as C 26:0-FA might affect cellular functions, thus leading to the pathogenesis of peroxisomal diseases, such as Zellweger syndrome and X-ALD.

Many cell surface proteins in mammalian cells are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The predominant form of mammalian GPI contains 1-alkyl-2-acyl phosphatidylinositol (PI), which is generated by lipid remodeling from diacyl PI. The conversion of diacyl PI to 1-alkyl-2-acyl PI occurs in the ER at the third intermediate in the GPI biosynthetic pathway. This lipid remodeling requires the alkyl-phospholipid biosynthetic pathway in peroxisome. Indeed, cells defective in dihydroxyacetone phosphate acyltransferase (DHAP-AT) or alkyl-DHAP synthase express only the diacyl form of GPI-anchored proteins. A defect in the alkyl-phospholipid biosynthetic pathway causes a peroxisomal disorder, rhizomelic chondrodysplasia punctata (RCDP), and defective biogenesis of peroxisomes causes Zellweger syndrome, both of which are lethal genetic diseases with multiple clinical phenotypes such as psychomotor defects, mental retardation, and skeletal abnormalities. Here, we report that GPI lipid remodeling is defective in cells from patients with Zellweger syndrome having mutations in the peroxisomal biogenesis factors PEX5, PEX16, and PEX19 and in cells from patients with RCDP types 1, 2, and 3 caused by mutations in PEX7, DHAP-AT, and alkyl-DHAP synthase, respectively. Absence of the 1-alkyl-2-acyl form of GPI-anchored proteins might account for some of the complex phenotypes of these two major peroxisomal disorders.

Human Pex16p, a peroxisomal membrane protein composed of 336 amino acids, plays a central role in peroxisomal membrane biogenesis. A nonsense mutation (R176ter) in the PEX16 gene has been reported in the case of only one patient (D-01) belonging to complementation group D of the peroxisome biogenesis disorders. We have now identified two patients belonging to group D (D-02 and D-03) whose fibroblasts were found to contain no peroxisomal membrane structure ghosts. Molecular analysis of the PEX16 gene revealed aberrant cDNA species lacking 65 bp, corresponding to exon 10 skipping caused by a splice site mutation (IVS10 + 2T -->C). Both patients, although unrelated, were homozygous for this mutation. This mutation changes the amino acid sequence starting from codon 298 and introduces a termination codon at codon 336. As a consequence, the cell's ability to membrane synthesis and protein import is disrupted, which implies that the changed C terminus of the Pex16p in these patients likely affects its function., ((C)2002 Elsevier Science (USA).)

Contiguous ABCD1 DXS1357E deletion syndrome ( CADDS) is a contiguous deletion syndrome involving the ABCD1 and DXS1357E/ BAP31 genes on Xq28. Although ABCD1 is responsible for X-linked adrenoleukodystrophy ( X-ALD), its phenotype differs from that of CADDS, which manifests with many features of Zellweger syndrome ( ZS), including severe growth and developmental retardation, liver dysfunction, cholestasis and early infantile death. We report here the fourth case of CADDS, in which a boy had dysmorphic features, including a flat orbital edge, hypoplastic nose, micrognathia, inguinal hernia, micropenis, cryptorchidism and club feet, all of which are shared by ZS. The patient achieved no developmental milestones and died of pneumonia at 8 months. Biochemical studies demonstrated abnormal metabolism of very long chain fatty acids, which was higher than that seen in X-ALD. Immunocytochemistry and Western blot showed the absence of ALD protein ( ALDP) despite the presence of other peroxisomal proteins. Pathological studies disclosed a small brain with hypomyelination and secondary hypoxic-ischemic changes. Neuronal heterotopia in the white matter and leptomeningeal glioneuronal heterotopia indicated a neuronal migration disorder. The liver showed fibrosis and cholestasis. The thymus and adrenal glands were hypoplastic. Array comparative genomic hybridization ( CGH) analysis suggested that the deletion was a genomic rearrangement in the 90-kb span starting in DXS1357E/ BACP31 exon 4 and included ABCD1, PLXNB3, SRPK3, IDH3G and SSR4, ending in PDZD4 exon 8. Thus, the absence of ALDP, when combined with defects in the B-cell antigen receptor associated protein 31 ( BAP31) and other factors, severely affects VLCFA metabolism on peroxisomal functions and produces ZS-like pathology. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisomal diseases are categorized into three large groups – peroxisome biogenesis disorders (PBD), single enzyme deficiencies (SED) and contiguous gene syndrome. Thirteen complementation groups and PEX genes responsible for all subgroups of PBD, plus 10 diseases and their responsible genes in SED have been identified. We have established a diagnostic system for peroxisomal diseases in Japan, and identified 45 Japanese patients with PBD, 12 patients with beta-oxidation enzyme deficiencies and more than 100 patients with adrenoleukodystrophy (ALD). It is important for effective therapy of the cerebral form of ALD to diagnose earlier after onset, and pre-symptomatic diagnosis should also be valuable. The division of diagnostic system into several specified centers of peroxisomal diseases in the whole world should be functional for overcoming these rare inherited neurometabolic diseases. [Copyright &y& Elsevier]

Peroxisome biogenesis factor 10 (PEX10) is involved in the import of peroxisomal matrix proteins, and the mutation of this gene causes 3 subtypes of peroxisome biogenesis disorders, namely Zellweger syndrome (severe), neonatal adrenoleukodystrophy (moderate) and an ataxic form (mild). Here, we report 3 siblings of the ataxic form with cerebellar ataxia, mild mental retardation, and 3 additional characteristic features: mydriasis, hyperreflexia and involuntary head movement. All 3 siblings are compound heterozygous for a previously reported mutation, c.2T > C (p.M1T), and a novel mutation, c.920G > A, causing a missense change (p.C307Y) located in the RING finger domain of PEX10 . The present cases suggest that these PEX10 mutations involve not only cerebellar but also more multiple nervous systems including pupillary autonomic, pyramidal and extrapyramidal systems. [ABSTRACT FROM AUTHOR]

Abstract Background Mutations in PEX16 cause peroxisome biogenesis disorder (PBD). Zellweger syndrome characterized by neurological dysfunction, dysmorphic features, liver disease and early death represents the severe end of this clinical spectrum. Here we discuss the diagnostic challenge of atypical PEX16 related PBD in 3 patients from highly inbred kindred and describe the role of specific metabolites analyses, fibroblasts studies, whole-exome sequencing (WES) and metabolomics profiling to establish the diagnosis. Methods and patients The proband is a 12-year-old male born to consanguineous parents. Despite normal development in the first year, regression and progressive spastic diplegia, poor coordination and dysarthria occurred thereafter. Patient 2 (3-year old female) and Patient 3 (19-month old female) shared similar clinical course with the proband. Biochemical studies on plasma and fibroblasts, WES and global metabolomics analyses were performed. Results Very-long-chain fatty acids analysis showed subtle elevations in C26 and C26/C22. Global Metabolomics-Assisted Pathway profiling was not remarkable. Immunocytochemical investigations on fibroblasts revealed fewer catalase and PMP70-containing particles indicating aberrant peroxisomal assembly. Complementation studies were inconclusive. WES revealed a novel homozygous variant in PEX16 (c.859C>T). The biochemical profiles of Patient 2 and Patient 3 were similar to the proband and the same genotype was confirmed. Conclusion This paper highlights the diagnostic challenge of PEX16 patients due to the widely variable clinical and biochemical phenotypes. It also emphasizes the important roles of combined biochemical assays with next generation sequencing techniques in reaching diagnosis in the context of atypical clinical presentations, subtle biomarker abnormalities and consanguinity. [ABSTRACT FROM AUTHOR]

Very long chain fatty acids (VLCFAs) and docosahexaenoic acid (DHA), phytanic acid, and plasmalogens are usually measured individually. A novel method for the screening of peroxisomal disorders, using gas chromatography/mass spectrometry (GC/MS), was developed. Saturated and unsaturated fatty acids, including VLCFAs and DHA, phytanic acid, and plasmalogen were detected by a selected ion monitoring-electron impact method, using 100 μl of serum or plasma. Methyl-esterification and extraction could be done in one tube, and data were obtained within 4 h. All patients with Zellweger syndrome (ZS), X-linked adrenoleukodystrophy (ALD), isolated deficiency of peroxisomal β-oxidation enzyme, and most ALD carriers showed increased VLCFA ratios, including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0. The ratio of DHA to palmitic acid (C16:0) and plasmalogen (measured as hexadecanal dimethyl acetal) to C16:0 in ZS patients was significantly lower than for the controls (P<0.001 for healthy high school students, P<0.05 for infants with other disorders). Plasmalogen was also decreased in patients with isolated deficiency of plasmalogen biosynthesis. Two of eight patients with ZS, two of four with RCDP, and all of three classical Refsum patients showed increased levels of phytanic acid. This method will simplify the screening for peroxisomal disorders. [Copyright &y& Elsevier]