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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]

Abstract: Peroxisomes are ubiquitous organelles in eukaryotic cells and surrounded by a single membrane, and undergo considerable changes in size, shape and number. Peroxisomal disorders are classified into two categories: peroxisome biogenesis disorders (PBDs) and single-enzyme deficiencies (SEDs). Morphologically aberrant peroxisomes called ‘peroxisomal ghosts’ in PBDs are well known, however, a morphological approach to the study of peroxisomes in SEDs has been rarely reported. Here, we investigated the morphology of peroxisomes in cultured fibroblasts from patients lacking peroxisomal beta-oxidation enzymes, including acyl-CoA oxidase (AOX) or D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-BP). Morphological analysis by immunofluorescence examination using an antibody against catalase revealed a smaller number of large peroxisomes in fibroblasts from these patients. Moreover, immunoelectron microscopy using an antibody against the 70-kDa peroxisomal membrane protein (PMP70) showed large peroxisomes with various horseshoe-shaped membrane structures. These results give an important clue to elucidating the division of peroxisomes and how peroxisomes change in size, shape, number and position within cells, which are subjects for future study. [Copyright &y& Elsevier]

Abstract Objective Early diagnosis is critical in achieving the best outcome following hematopoietic stem cell transplantation (HSCT) for X-linked adrenoleukodystrophy (X-ALD). We used a questionnaire to gather detailed clinical information and information regarding the anxieties of patients' families using the registry system for X-ALD. Methods We and the patients' families established the registry system for X-ALD in Japan. We created a questionnaire and distributed it to the patients' families. Results Questionnaire data from 28 patients were collected. The median age at enrollment was 14.5 years. The most common type of X-ALD was the childhood cerebral form (22 patients, 78.6%). The median age at symptom onset was 7.4 years. Frequently reported initial observations were behavior or character changes (46.4%), gait disturbances (42.9%), strabismus (39.3%), reduced academic ability (32.1%), failing vision (21.4%), a positive family history (21.4%), clumsiness (17.9%), hearing disturbances (17.9%), convulsions (10.7%), and suspected psychiatric disorders (10.7%). The median duration from symptom onset to diagnosis was 12 months. The families of 12 patients (42.9%) with X-ALD who received HSCT were satisfied regardless of its effectiveness. Common concerns of patients' families were worries regarding heritability of X-ALD (78.6%), present symptoms (57.1%), frequent hospital visits (42.9%), problems at school or work (42.9%), economic issues (35.7%), and limited information regarding X-ALD (32.1%). Conclusion This is the first study clarifying the clinical characteristics of X-ALD and the concerns of patients' families using the registry system. Investigation of rare diseases using registry systems is very valuable for the understanding of such conditions. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Abstract: Changes in the molecular species of lipids associated with peroxisomal d-bifunctional protein (d-BP) deficiency were investigated in cerebral tissues to elucidate the pathological mechanisms underlying this disorder. Total phospholipids in the gray and white matters of the patient''s brain were decreased to approximately 73% and 50% of control levels, respectively, and profound declines in myelin lipids, i.e. galactosyl ceramide and sulfatides, indicated dysmyelination in our patient with d-BP deficiency. Although the total ganglioside amounts in the gray and white matter of this patient''s brain were also decreased to 61% and 37% of control levels and GM1 in the white matter was 20% of the control level, the relative amounts of GM2 in both the gray and the white matter of this patient''s brain were increased in comparison to those in the control, indicating altered metabolism of gangliosides. In addition, among molecular species of phospholipids, plasmalogen-type and polyunsaturated fatty acid-containing phosphatidylethanolamine were characteristically decreased in the patient''s gray matter. These alterations in the molecular species of brain lipids may affect sensitivity to oxidative stress and the membrane fluidity of neural cells, thereby producing the brain pathology of d-BP deficiency. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to assess mental health in parents of patients with the childhood cerebral form of X-linked adrenoleukodystrophy (CCALD) and to investigate factors relating to psychological problems in order to improve clinical management and quality of life. Sixteen fathers and 21 mothers of patients with CCALD completed a battery of psychological examinations including the Beck Depression Inventory second edition (BDI-II), the General Health Questionnaire 60 (GHQ60), and the State–Trait Anxiety Inventory (STAI). Three fathers and 11 mothers showed high scores on the BDI-II, suggesting that they were in a depressive state. Depression in the mothers was serious as compared with previous reports. Six fathers and 11 mothers were considered to be in a state of neurosis, according to the results of the GHQ60. Four fathers and 8 mothers showed high levels of anxiety on the STAI. Health and social status of the mothers correlated with their mental health, and younger mothers with young patients tended to be more depressed. Thus, parents of patients with CCALD have a high risk of depression and neurosis. Understanding the mental state of these parents and improvements in the social support system including mental counseling, home nursing care, supports in workplace and community are necessary to prevent and treat psychological problems. Especially, early intervention for mental health problems should be provided for younger mothers with few years since the child’s diagnosis. [Copyright &y& Elsevier]

Abstract: The natural history of X-linked adrenoleukodystrophy (ALD) was investigated, using a nation-wide retrospective study based on a questionnaire survey. The data on 145 patients, including 46 patients with the childhood cerebral form, 39 with adrenomyeloneuropathy (AMN), 33 with the adult cerebral form, 14 with the adolescent form and 13 with the olivo-ponto-cerebellar (OPC) form, were analyzed. Initial symptoms of the childhood cerebral form were intellectual (n=16) and visual (n=11) disturbances, whereas those of AMN were gait (n=37) and sensory (n=3) disturbances; the adult cerebral form, psychic (n=19) and gait (n=11) disturbances; the adolescent form, visual n=5) and gait (n=4) disturbances; and the OPC form, gait (n=9) disturbance. Patients with onset under the age of 8 years progressed more rapidly than those over 8 years old. Visual, hearing, gait and swallowing disturbances progressed more slowly in the older group. About half of AMN patients showed cerebral involvement about 10 years after onset. Patients with the OPC form also showed a similar progression. A Kaplan–Meier plot clarified the characteristic pattern of progression of neurological symptoms in each phenotype. These finding will improve the understanding of the natural history of X-linked ALD and will provide a basis for the evaluation of specific treatment for X-linked ALD. [Copyright &y& Elsevier]

Childhood cerebral adrenoleukodystrophy (CCALD) is the most common phenotype of adrenoleukodystrophy (ALD) and is characterized by the progression of intellectual, psychic, visual, and gait disturbances. Progression of this intractable disease can only be prevented by hematopoietic stem cell transplantation during the early stages of the disease. The aim of this study was to clinically evaluate children with CCALD who have visual symptoms to enable early diagnosis. We enrolled 41 Japanese children with CCALD who had visual symptoms. We retrospectively analyzed age of onset, past medical history, initial symptoms, visual symptoms and findings on brain magnetic resonance imaging. The median age of disease onset was 7 years (range 5–10 years). The most common visual symptom was strabismus (n = 22). There was only one patient with the triad of symptoms of Balint's syndrome. Seventeen patients had incomplete Balint's syndrome and showed one or two of the triad of symptoms. Almost all patients with complete or incomplete Balint's syndrome showed bilateral parieto-occipital white matter lesions. CCALD could develop into Balint's syndrome, especially the incomplete form. Therefore, CCALD should be considered when boys show new symptoms, including lack of eye contact or bumping into objects. [ABSTRACT FROM AUTHOR]

Peroxisomal acyl-CoA oxidase (ACOX1) deficiency is a rare autosomal recessive single enzyme deficiency characterized by hypotonia, seizures, failure to thrive, developmental delay, and neurological regression starting from approximately 3 years of age. Here, we report two siblings with ACOX1 deficiency born to non-consanguineous Japanese parents. They showed mild global developmental delay from infancy and began to regress at 5 years 10 months and 5 years 6 months of age respectively. They gradually manifested with cerebellar ataxia, dysarthria, pyramidal signs, and dysphasia. Brain MRI revealed T2 high-intensity areas in the cerebellar white matter, bilateral middle cerebellar peduncle, and transverse tracts of the pons, followed by progressive atrophy of these areas. Intriguingly, the ratios of C24:0, C25:0, and C26:0 to C22:0 in plasma, which usually increase in ACOX1 deficiency were within normal ranges in both patients. On the other hand, whole exome sequencing revealed novel compound heterozygous variants in ACOX1 : a frameshift variant (c.160delC:p.Leu54Serfs*18) and a missense variant (c.1259 T > C:p.Phe420Ser). The plasma concentration of individual very long chain fatty acids (C24:0, C25:0, and C26:0) was elevated, and we found that peroxisomes in fibroblasts of the patients were larger in size and fewer in number as previously reported in patients with ACOX1 deficiency. Furthermore, the C24:0 β-oxidation activity was dramatically reduced. Our findings suggest that the elevation of individual plasma very long chain fatty acids concentration, genetic analysis including whole exome analysis, and biochemical studies on the patient's fibroblasts should be considered for the correct diagnosis of ACOX1 deficiency. [ABSTRACT FROM AUTHOR]

Biallelic variants in POLR3A encoding the largest subunit of RNA polymerase III cause POLR3-related (or 4H) leukodystrophy characterized by neurologic dysfunction, abnormal dentition, endocrine abnormalities and ocular abnormality. Recently, whole-exome sequencing enabled the discovery of POLR3A variants in cases lacking diffuse hypomyelination, the principal MRI phenotype of POLR3-related leukodystrophy. Homozygous c.1771-6C > G variants in POLR3A were recently suggested to cause striatal and red nucleus involvement without white matter involvement. Case report: Here, we report three cases in two families with biallelic POLR3A variants. We identified two sets of compound heterozygous variants in POLR3A , c.1771-6C > G and c.791C > T, p.(Pro264Leu) for family 1 and c.1771-6C > G and c.2671C > T, p.(Arg891*) for family 2. Both families had the c.1771-6C > G variant, which led to aberrant mRNA splicing. Neuropsychiatric regression and severe intellectual disability were identified in three patients. Two cases showed dystonia and oligodontia. Notably, characteristic bilateral symmetric atrophy and abnormal signal of the striatum without diffuse white matter signal change were observed in brain MRI of all three individuals. Striatum abnormalities may be another distinctive MRI finding associated with POLR3A variants, especially in cases including c.1771-6C > G variants and our cases can expand the phenotypic spectrum of POLR3A -related disorders. [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]

Abstract: We identified the first patient with infantile Refsum disease (IRD), a milder phenotype of peroxisome biogenesis disorder (PBD) caused by a mutated PEX3, and investigated the clinical, molecular and cellular characterization in this patient. The patient presented psychomotor regression, late-onset leukodystrophy, peripheral neuropathy, hearing impairment, a renal cyst, and renal hypertension and survived until the age of 36. Furthermore, fibroblasts from the patient indicated a mosaic pattern of catalase-positive particles (peroxisomes) and numerous peroxisomal membrane structures. Molecular analysis was homozygous for the D347Y mutation and reduced gene expression of PEX3 which encodes a peroxisomal membrane protein, pex3p, involved in peroxisome assembly at the early stage of peroxisomal membrane vesicle formation, therefore, patients with a mutated PEX3 gene have been reported to have only a severe phenotype of Zellweger syndrome and no or less peroxisomal remnant membrane structure. This is not only a newly identified milder PBD caused by a mutated PEX3 gene but also the first report of a Japanese patient with IRD who had not been diagnosed until over 30years of age, which suggests there must be more variant PBD in patients with degenerative neurologic disorder, and to bring them to light is necessary. [Copyright &y& Elsevier]

Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macromolecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. Because of the high sensitivity achieved by the present proteomics technology, the purity of sampies to be analyzed is important for the interpretation of the results obtained. In the present study, peroxisomes isolated from rat liver by usual cell fractionation were further purified by immunoisolation using a specific antibody raised against a peroxisomal membrane protein, PMP70. The isolated peroxisomes were analyzed by SDS-PAGE combined with liquid chromatography/mass spectrometry. Altogether 34 known peroxisomal proteins were identified in addition to several mitochondrial and microsomal proteins. Some of the latter may reside in the peroxisomes as well. Analysis of membrane fractions identified all known peroxins except for Pex7. Two new peroxisomal proteins of unknown function were of high abundance. One is a bi-functional protein consisting of an aminoglycoside phosphotransferase-domain and an acyl-CoA dehydrogenase domain. The other is a newly identified peroxisome-specific isoform of Lon protease, an ATP-dependent protease with chaperonelike activity. The peroxisomal localization of the protein was confirmed by immunological techniques. The peroxisome-type Lon protease, which is distinct from the mitochondrial isoform, may play an important role in the peroxisomal biogenesis. [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]

Since the first report by Brenner et al. of mutations in the glial fibrillary acidic protein (GFAP) gene in patients with Alexander disease, several molecular genetic studies have been performed in different ethnic groups. We previously reported a Japanese patient with a mutation, R239C, which is identical to one commonly found in American patients. Here we have analyzed four additional Japanese patients by screening for known mutations or, if no known mutation was found, by sequencing of all exons of the GFAP gene. We detected three missense mutations; one was a novel mutation, R79L, and two were previously reported mutations, R239C and R79C. All of our patients were heterozygous for their mutations. Together with the novel mutation, R79L, four different nucleotide changes altering the R79 residue have been reported, implying that any alternation of this arginine residue can give the GFAP protein a dominant negative effect, leading to accumulation of GFAP as Rosenthal fibers. We conclude that molecular genetic analysis of the GFAP gene is feasible for antemortem diagnosis of Alexander disease in Japanese patients. [Copyright &y& Elsevier]