Molecular and clinical findings and diagnostic flowchart of peroxisomal diseases.

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.

Peroxisomal disorders with infantile seizures.

Peroxisomes are organelles responsible for multiple metabolic pathways including the biosynthesis of plasmalogens and the oxidation of branched-chain as well as very-long-chain fatty acids (VLCFAs). Peroxisomal disorders (PDs) are heterogeneous groups of diseases and affect many organs with varying degrees of involvement. Even pathogenetically distinct PDs share some common symptoms. However, several PDs have uniquely characteristic clinical findings. The durations of survival in PDs are also variable. Infants with PDs are usually presented with developmental delay, visual and hearing impairment. Generalized hypotonia is present in severe cases. Epileptic seizures are also a common characteristic of patients with certain PDs. Nonetheless, the classification and evolution of epilepsy in PDs have not been elucidated in detail. Here, we review the relevant literatures and provide an overview of PDs with particular emphasis on the characteristics of seizures in infants.

Molecular and clinical aspects of peroxisomal diseases.

Peroxisomal diseases, an expanding group of inborn errors of metabolism, can be classified into three categories--peroxisome biogenesis disorders (PBDs), single peroxisomal enzyme deficiencies, and contiguous gene syndrome. PBDs comprise 13 complementation groups and their responsible genes have been identified, including our newly identified group with a PEX14 defect. We have established a diagnostic system of peroxisomal diseases in Japan, and have identified 40 Japanese with PBDs, 11 patients with beta-oxidation enzyme deficiencies and more than 100 patients with adrenoleukodystrophy. Further study of and enlightenment on peroxisomal diseases is necessary to overcome these disorders.

Clinical and biochemical spectrum of D-bifunctional protein deficiency.

OBJECTIVE: D-bifunctional protein deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. Although case reports and small series of patients have been published, these do not give a complete and balanced picture of the clinical and biochemical spectrum associated with this disorder. METHODS: To improve early recognition, diagnosis, prognosis, and management of this disorder and to provide markers for life expectancy, we performed extensive biochemical studies in a large cohort of D-bifunctional protein-deficient patients and sent out questionnaires about clinical signs and symptoms to the responsible physicians. RESULTS: Virtually all children presented with neonatal hypotonia and seizures and died within the first 2 years of life without achieving any developmental milestones. However, within our cohort, 12 patients survived beyond the age of 2 years, and detailed information on 5 patients with prolonged survival (> or =7.5 years) is provided. INTERPRETATION: Biochemical analyses showed that there is a clear correlation between several biochemical parameters and survival of the patient, with C26:0 beta-oxidation activity in cultured skin fibroblasts being the best predictive marker for life expectancy. Remarkably, three patients were identified without biochemical abnormalities in plasma, stressing that D-bifunctional protein deficiency cannot be excluded when all peroxisomal parameters in plasma are normal.

Mutations in novel peroxin gene PEX26 that cause peroxisome-biogenesis disorders of complementation group 8 provide a genotype-phenotype correlation.

The human disorders of peroxisome biogenesis (PBDs) are subdivided into 12 complementation groups (CGs). CG8 is one of the more common of these and is associated with varying phenotypes, ranging from the most severe, Zellweger syndrome (ZS), to the milder neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD). PEX26, encoding the 305-amino-acid membrane peroxin, has been shown to be deficient in CG8. We studied the PEX26 genotype in fibroblasts of eight CG8 patients--four with the ZS phenotype, two with NALD, and two with IRD. Catalase was mostly cytosolic in all these cell lines, but import of the proteins that contained PTS1, the SKL peroxisome targeting sequence, was normal. Expression of PEX26 reestablished peroxisomes in all eight cell lines, confirming that PEX26 defects are pathogenic in CG8 patients. When cells were cultured at 30 degrees C, catalase import was restored in the cell lines from patients with the NALD and IRD phenotypes, but to a much lesser extent in those with the ZS phenotype, indicating that temperature sensitivity varied inversely with the severity of the clinical phenotype. Several types of mutations were identified, including homozygous G89R mutations in two patients with ZS. Expression of these PEX26 mutations in pex26 Chinese hamster ovary cells resulted in cell phenotypes similar to those in the human cell lines. These findings confirm that the degree of temperature sensitivity in pex26 cell lines is predictive of the clinical phenotype in patients with PEX26 deficiency.

Human peroxisomal disorders.

Peroxisomes are single membrane-bound cell organelles performing numerous metabolic functions. The present article aims to give an overview of our current knowledge about inherited peroxisomal disorders in which these organelles are lacking or one or more of their functions are impaired. They are multiorgan disorders and the nervous system is implicated in most. After a summary of the historical names and categories, each having distinct symptoms and prognosis, microscopic pathology is reviewed in detail. Data from the literature are added to experience in the authors' laboratory with 167 liver biopsy and autopsy samples from peroxisomal patients, and with a smaller number of chorion samples for prenatal diagnosis, adrenal-, kidney-, and brain samples. Various light and electron microscopic methods are used including enzyme- and immunocytochemistry, polarizing microscopy, and morphometry. Together with other laboratory investigations and clinical data, this approach continues to contribute to the diagnosis and further characterization of peroxisomal disorders, and the discovery of novel variants. When liver specimens are examined, three main groups including 9 novel variants (33 patients) are distinguished: (1) absence or (2) presence of peroxisomes, and (3) mosaic distribution of cells with and without peroxisomes (10 patients). Renal microcysts, polarizing trilamellar inclusions, and insoluble lipid in macrophages in liver, adrenal cortex, brain, and in interstitial cells of kidney are also valuable for classification. On a genetic basis, complementation of fibroblasts has classified peroxisome biogenesis disorders into 12 complementation groups. Peroxisome biogenesis genes (PEX), knock-out-mice, and induction of redundant genes are briefly reviewed, including some recent results with 4-phenylbutyrate. Finally, regulation of peroxisome expression during development and in cell cultures, and by physiological factors is discussed.

Phenotypic variability (heterogeneity) of peroxisomal disorders.

Peroxisomes perform a multitude of biosynthetic and catabolic functions, many of which are related to lipid metabolism. Peroxisomal disorders result either from deficiency of a single peroxisomal enzyme or protein, or from a defect in the complex mechanism of peroxisomal biogenesis, resulting in deficiency of several or multiple peroxisomal functions. These can be assessed by a battery of biochemical assays, enabling a biochemical phenotype to be defined that is specific and diagnostic for each of the peroxisomal disorders. Some peroxisomal disorders have unique and specific clinical phenotypes, which may be diagnostic. Others share patterns of clinical abnormalities (particularly neurological dysfunction, craniofacial dysmorphism, skeletal defects, sensory deafness, retinopathy) consistent with defined clinical phenotypes, but with considerable overlap and heterogeneity. To a certain extent, the clinical features of a particular disorder reflect the accumulation or deficiency of specific metabolites. Thus, the same clinical phenotypes may be caused by both single enzyme defects and PBDs. Furthermore, the same defect may present with different clinical phenotypes. In general, the severity of the clinical phenotype correlates with the degree of biochemical dysfunction. The clinical heterogeneity of peroxisomal disorders constitutes a diagnostic challenge demanding a high index of suspicion on the clinician's part.

Peroxisomes in dermatology. Part I.

BACKGROUND: Peroxisomes are small cellular organelles that were almost ignored for years because they were believed to play only a minor role in cellular functions. However, it is now known that peroxisomes play an important role in regulating cellular proliferation and differentiation as well as in the modulation of inflammatory mediators. In addition, peroxisomes have broad effects on the metabolism of lipids, hormones, and xenobiotics. Through their effects on lipid metabolism, peroxisomes also affect cellular membranes and adipocyte formation, as well as insulin sensitivity, and peroxisomes play a role in aging and tumorigenesis through their effects on oxidative stress. OBJECTIVE: To review genetically determined peroxisomal disorders, especially those that particularly affect the skin, and some recent information on the specific genetic defects that lead to some of these disorders. In addition, we present some of the emerging knowledge of peroxisomal proliferator activator receptors (PPARs) and how ligands for these receptors modulate different peroxisomal functions. We also present information on how the discovery of PPARs, and the broad and diverse group of ligands that activate these members of the superfamily of nuclear binding transcription factors, has led to development of new drugs that modulate the function of peroxisomes. CONCLUSION: PPAR expression and ligand modulation within the skin have shown potential uses for these ligands in a number of inflammatory cutaneous disorders, including acne vulgaris, cutaneous disorders with barrier dysfunction, cutaneous effects of aging, and poor wound healing associated with altered signal transduction, as well as for side effects induced by the metabolic dysregulation of other drugs.

The peroxin Pex6p gene is impaired in peroxisomal biogenesis disorders of complementation group 6.

Human genetic peroxisomal biogenesis disorders (PBDs), such as Zellweger syndrome, comprise 13 different complementation groups (CGs). Eleven peroxin genes, termed PEXs, responsible for PBDs have been identified, whereas pathogenic genes for PBDs of 2CGs, CG-A (the same CG as CG8 in the United States and Europe) and CG6, remained unidentified. We herein provide several lines of novel evidence indicating that PEX6, the pathogenic gene for CG4, is impaired in PBD of CG6. Expression of PEX6 restored peroxisome assembly in fibroblasts from a CG6 PBD patient. This patient was a compound heterozygote for PEX6 gene alleles. Accordingly, by merging CG6 with CG4, human PBDs are now classified into 12CGs.

Hepatic peroxisomes in isolated hyperpipecolic acidaemia: evidence supporting its classification as a single peroxisomal enzyme deficiency.

Hyperpipecolic acidaemia is still regarded as a peroxisomal assembly deficiency. The enzyme responsible for the accumulation of pipecolic acid is located in the peroxisomes in man. We studied the appearance and alterations of peroxisomes in liver biopsy material from three unrelated children suffering from isolated hyperpipecolic acidaemia, in which only the metabolism of pipecolic acid is disturbed, using light and electron microscopy after cytochemical staining for visualisation of peroxisomes. Morphometric results showed the presence of normal-sized to small peroxisomes, an increase in number and abnormally shaped organelles, suggesting enhancement of metabolic efficiency. In one case enlarged organelles were observed. Skin fibroblasts were studied in all patients: their peroxisomes appeared to be normal. The obvious presence of peroxisomes in isolated HPA indicates that this disorder should be classified as a single peroxisomal enzyme deficiency.

Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins.

We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.

Peroxisomal disorders: clinical, biochemical, and molecular aspects.

Peroxisomes are subcellular organelles catalyzing a number of indispensable functions in cellular metabolism. The importance of peroxisomes in man is stressed by the existence of an expanding group of genetic diseases in which there is an impairment in one or more peroxisomal functions. Much has been learned in recent years about these functions and many of the enzymes involved have been characterized, purified and their cDNAs cloned. This has allowed resolution of the enzymatic and molecular basis of many of the single peroxisomal enzyme deficiencies. Similarly, the molecular basis of the peroxisome biogenesis disorders is also being resolved rapidly thanks to the successful use of CHO as well as yeast mutants. In this paper we will provide an overview of the peroxisomal disorders with particular emphasis on their clinical, biochemical and molecular characteristics.

Paul Dyken Lecture of the Southern Pediatric Neurology Society. Inherited neurodegenerative disease: the evolution of our thinking.

The past 3 decades have witnessed impressive progress in our understanding of inherited neurometabolic diseases, promoted by the rapid development and application of molecular genetic strategies. Such progress has required the juxtaposition of clinical evaluations and basic science techniques. The central role of careful and complete assessment of affected children cannot be overemphasized and in no way has been diminished by technologic advances. Indeed, enhanced clinical and laboratory evaluations have led to important conceptual advances. Molecular genetics has elucidated those disorders with known metabolic defects through functional cloning and explained the variability of disease expression based on specific mutational events. Alternatively, positional cloning has identified molecular defects for those disorders with clear phenotypic patterns, but lacking a defined metabolic abnormality. Regarding heterogeneous expression, disorders with clearly different phenotypes can arise from different mutations within the same gene. The multiple variants of beta-hexosaminidase deficiency (Tay-Sachs disease) are, arguably, the best examples. Conversely, disorders with similar phenotypes are explainable by quite different mutational events. In addition, the identification of specific diseases exhibiting both biochemical abnormalities and disturbed organogenesis has blurred conventional dogma regarding separation of genetic disorders into strict metabolic and structural categories. Disorders of peroxisomal function and the neuronal ceroid lipofuscinoses are prototypes for the points noted above and raise important issues regarding our approaches to children with these disorders. These issues include a high index of suspicion for an inherited neurometabolic disease and an open mind to possible interrelations with other known and seemingly dissimilar conditions.

[Peroxisomal disorders: classification and overview of biochemical abnormalities]. / Enfermedades peroxisomales: clasificación y descripción de las anomalías bioquímicas.

The peroxisomal disorders are subdivided into two major categories: those in which the organelle is not formed normally (disorders of peroxisome biogenesis), and those that are associated with defects of single peroxisomal proteins. The Zellweger cerebrohepatorenal syndrome is the prototype of the peroxisome biogenesis disorders. It has been shown to be due to defective import of proteins into the organelle. Ten distinct molecular defects can lead to the failure of import.

[Molecular analysis of peroxisomal disorders].

Peroxisome biogenesis disorders (PBD) include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD). They are classified into ten complementation groups. Five pathogenic genes have been identified using different model systems of peroxisome deficient mutants. PAF-1 and 2 were identified from CHO mutants and were responsible genes for PBD group F and C. Human PEX 5, 12 and 1, responsible genes for group 2, 3 and 1, respectively, were cloned by homology search between yeast PEX genes and human genes on the cDNA data base. Adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder, shows phenotypic heterogeneity. Its responsible gene was cloned by positional cloning. It encodes a 75 kDa peroxisomal membrane protein (ALDP) that is a member of the ATP-binding cassette transporter family. There are about 120 different mutations including missense, nonsense and splice mutations, as well as insertions and deletions of a few base pairs. There is no correlation between the clinical phenotype and the ALDP gene mutation. Recently, animal models have been produced by targeted mutation of the PBD and ALD genes. The mouse model should facilitate researches on PBD and ALD, especially those on regulatory factors of their phenotypic heterogeneity and on new therapeutic approaches.

Isolation and characterization of peroxisome-deficient Chinese hamster ovary cell mutants representing human complementation group III.

We made use of the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method and isolated peroxisome-deficient mutant cells. TKa cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1) were used to avoid frequent isolation of the Z65-type, PEX2-defective mutants. P9OH/UV-resistant cell colonies were examined for the intracellular location of catalase, a peroxisomal matrix enzyme, by immunofluorescence microscopy and using anti-catalase antibody. As six mutant cell clones showed cytosolic catalase, there was likely to be a deficiency in peroxisome assembly. These mutants also showed the typical peroxisome assembly-defective phenotype, including significant decrease of dihydroxyacetonephosphate acyltransferase, the first step key enzyme in plasmalogen synthesis, and loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. By transfection of Pex2p and Pex6p (formerly PAF-2) cDNAs and cell fusion analysis between the CHO cell mutants, two mutants, ZP104 and ZP109, were found to belong to a novel complementation group. Further complementation analysis using fibroblasts from patients with peroxisome biogenesis disorders revealed that the mutants belonged to human complementation group III. Taken together, ZP104 and ZP109 are in a newly identified fifth complementation group in CHO mutants reported to date and represent the human complementation group III.

Biochemistry of peroxisomes in health and disease.

The ubiquitous distribution of peroxisomes and the identification of a number of inherited diseases associated with peroxisomal dysfunction indicate that peroxisomes play an essential part in cellular metabolism. Some of the most important metabolic functions of peroxisomes include the synthesis of plasmalogens, bile acids, cholesterol and dolichol, and the oxidation of fatty acids (very long chain fatty acids > C22, branched chain fatty acids (e.g. phytanic acid), dicarboxylic acids, unsaturated fatty acids, prostaglandins, pipecolic acid and glutaric acid). Peroxisomes are also responsible for the metabolism of purines, polyamines, amino acids, glyoxylate and reactive oxygen species (e.g. O-2 and H2O2). Peroxisomal diseases result from the dysfunction of one or more peroxisomal metabolic functions, the majority of which manifest as neurological abnormalities. The quantitation of peroxisomal metabolic functions (e.g. levels of specific metabolites and/or enzyme activity) has become the basis of clinical diagnosis of diseases associated with the organelle. The study of peroxisomal diseases has also contributed towards the further elucidation of a number of metabolic functions of peroxisomes.

[Peroxisomal hereditary diseases]. / Peroxizómové dedicné ochorenia.

Nearly two tens of diseases are known to be caused by impairment of several metabolic functions of peroxisomes, or by deficiency in individual peroxisomal enzymes. With the exception of X-bound adrenoleukodystrophy, all diseases are based on autosomally recessive type of inheritance and a majority of them are characteristic by specific neurologic symptoms. The group of diseases in which patients develop a generalised loss of peroxisomal functions includes: Zellweger's cerebro-hepato-renal syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, hyperpipecolic acidaemia. Other diseases, such as rhizomelic chondrodysplasia punctata and Zellweger-like syndrome are accompanied by a deficiency in several enzymatic activities. X-bound adrenoleukodystrophy, pseudo-Zellweger's syndrome, hyperoxaluria 1, adult form of Refsum's disease and acatalasaemia are peroxisomal diseases with a deficiency of a single enzyme. In clinically most severe diseases (generalised loss of peroxisomal functions), the impairment of peroxisomal biogenesis is caused assumedly due to the defect in some of the peroxisomal membrane proteins. The biochemical findings are brought about by insufficiency in such metabolic functions as oxidation of fatty acids with very long chains, oxidation of the phytanic and pipecolic acids, synthesis of cholesterol, bile salts and plasmalogenes. Rhizomelic chondrodysplasia punctata and Zellweger's syndrome are more moderate forms which are dominantly biochemically manifestant by an impairment in the synthesis of plasmalogenes. Among the diseases characterised by a deficiency in individual peroxisomal enzymes, most frequent is the X-bound andrenoleukodystrophy which has several clinical phenotypes manifestant in childhood, as well as a clinically less severe form manifestant in adulthood-adrenomyeloneuropathy. The diagnosis of peroxisomal diseases is performed by use of a wide range of methods (morphological, biochemical, immunochemical and molecular genetic examinations) which enable both postnatal and prenatal diagnostics. (Tab. 1, Ref. 104.)