review September/October 2001 ⅐ Vol. 3 ⅐ No. 5 Genetic and molecular bases of peroxisome biogenesis disorders Yasuyuki Suzuki, MD, PhD1,2, Nobuyuki Shimozawa, MD, PhD2, Tadao Orii, MD, PhD3, Toshiro Tsukamoto, MD, PhD4, Takashi Osumi, PhD4, Yukio Fujiki, PhD5, and Naomi Kondo, MD, PhD2 Peroxisomes are ubiquitous organelles present in almost all period, psychomotor retardation, facial dysmorphism such as eukaryotic cells and have a number of important metabolic high forehead and large fontanelle, hepatomegaly with pro- pathways. They were discovered in 1954 and named peroxi- longed jaundice and liver dysfunctions, calcific stippling of the somes in 1965.1 Peroxisomes have their own system of fatty patella, renal cortical microcysts, retinal degeneration, and acid ␤-oxidation.2,3 This system catalyzes a wide variety of sub- congenital heart disease, and usually die during the early infan- strates, including very long chain fatty acids (VLCFAs) such as tile period. Neonatal adrenoleukodystrophy is a phenotype lignoceric acid (C24:0) and cerotic acid (C26:0), bile acid in- with less severe clinical findings. Patients with NALD gradually termediates such as trihydroxycholestanoic acid (THCA) and develop, but then regression occurs, and patients usually die dihydroxycholestanoic acid (DHCA), and branched chain during the late infantile period. Infantile Refsum disease is fatty acids like pristanic acid and phytanic acid. Plasmalogens characterized by hearing impairment, retinal degeneration, are a special group of phospholipids, which are an important and mild psychomotor retardation, and patients usually sur- constituent of the brain and require peroxisomes for their syn- vive longer.14 thesis.4 Glyoxylate is a precursor of oxalate and is metabolized Defective neuronal migration is the main pathological find- to glycine by alanine-glyoxylate aminotransferase.5 Peroxi- ing in ZS.15 Polymicrogyria, pachygyria, heterotopia of cere- somes are also considered to play roles in the synthesis of do- bral neurons and Purkinje cells in the cerebellum, dysplasia of cosahexaenoic acid (DHA)6 and cholesterol.7 Catalase, a olivary complex, and hypomyelination are also characteristic marker enzyme of peroxisomal matrix, plays an important role findings. Architecture of cerebral cortex is disorganized and in the degradation of hydrogen peroxide.1 abnormally thick. The cerebellar Purkinje layer is irregular, Inborn errors of peroxisomes are classified into two catego- and heterotopic Purkinje cells are seen in the cerebellar white ries: peroxisome biogenesis disorders (PBDs) and single en- matter. The olivary nucleus is thick and poorly convoluted. zyme deficiencies (Table 1). Generalized PBDs include Zell- The NALD brain shows demyelination. Lamellar inclusion weger’s cerebro-hepato-renal syndrome (ZS),8 the most severe bodies, which consist of cholesterol esters of VLCFAs, are ob- phenotype; neonatal adrenoleukodystrophy (NALD),9 the in- served in macrophages and adrenocortical cells. termediate; and infantile Refsum disease (IRD),10 the least se- Biochemical abnormalities of PBDs are as follows: accumu- vere phenotype. Rhizomelic chondrodysplasia punctata lation of VLCFAs (C24:0, C25:0, C26:0), intermediate sub- (RCDP)11 is a partial PBD. Deficiency of peroxisomes in ZS stances of bile acid synthesis (THCA, DHCA) and branched was first identified in 1973,12 then the accumulation of VL- chain fatty acid (pristanic and phytanic acid); deficiency of CFAs was first reported in 1982.13 These PBDs are caused by a plasmalogens and DHA; and absent or severely decreased per- defect in PEX genes, which encode peroxins, proteins neces- oxisomes. All phenotypes of PBDs show these findings; how- sary for peroxisome biogenesis and import of peroxisomal ever, patients with milder phenotypes tend to have less severe proteins. abnormalities.14 Peroxisomes are morphologically absent or severely reduced in hepatocytes (Fig. 1C) or fibroblasts (Fig. CLINICAL, PATHOLOGICAL, AND BIOCHEMICAL 1D). Peroxisomal ghosts, which are remnant membranous FINDINGS OF PBDs structures, can be detected in many PBD cell lines when the cells are stained with anti-PMP70,16 a major peroxisomal Zellweger syndrome was first reported in 1964 as an autoso- membrane protein. However, some PBD cell lines lack 8 mal recessive disease with multiple congenital malformations. ghosts,17 which suggests that biogenesis of peroxisomal mem- Patients with ZS manifest severe hypotonia from the neonatal brane itself is disordered. Peroxisomes also show another in- teresting phenomenon: temperature sensitivity (ts) of peroxi- 1 2 From the Medical Education Development Center and Department of Pediatrics, Gifu some biogenesis.18 That is the biogenesis of peroxisomes and 3 University School of Medicine, Gifu; Faculty of Human Welfare, Chubu Gakuin University, biochemical defects in the cells from patients with milder phe- Seki; 4Department of Life Science, Himeji Institute of Technology, Hyogo; and 5Department of Biology, Graduate School of Science, Kyushu University, Fukuoka, Japan. notypes such as NALD and IRD are restored at the lower tem- Њ Yasuyuki Suzuki, MD, Medical Education Development Center, Gifu University School of perature, 30 C, whereas the biogenesis is not restored in the Medicine, Tsukasa-machi 40, Gifu 500-8705, Japan. severe Zellweger cells at 30ЊC. This phenomenon is useful for Received: November 21, 2000. predicting the clinical severity and for investigating peroxiso- Accepted: June 19, 2001. mal biogenesis. 372 Genetics IN Medicine Peroxisome biogenesis disorders Table 1 be formed in multinuclear cells a few days after cell fusion. An Peroxisomal disorders international collaborative study has led to the identification of A. Peroxisome biogenesis disorders (PBDs) 12 groups at present (Table 2).17 ZS, NALD, and IRD belong to 1. Zellweger syndrome (ZS) 11 groups. RCDP is genetically heterogeneous and belongs to one PBD group and the single enzyme deficiency group. Pa- 2. Neonatal adrenoleukodystrophy (NALD) tients with dihydroxyacetone phosphate (DHAP) acyltrans- 3. Infantile Refsum disease (IRD) ferase deficiency and alkyl-DHAP synthase deficiency also 4. Rhizomelic type chondrodysplasia punctata (RCDP) manifest RCDP phenotype. Group 1(E) is the largest group, 5. Zellweger-like syndrome and more than half the number of patients belong to this B. Isolated enzyme deficiencies group. Groups D, G, and J lack peroxisomal ghosts, and these groups contain only ZS, the most severe phenotype. At present, 1. Adrenoleukodystrophy (ALD) 11 of the genes associated with the different complementation 2. Acyl-CoA oxidase deficiency groups have been identified. These genes are called PEX genes, 3. D-Bifunctional protein deficiency and the gene products are called Pexp (peroxins). 4. 3-Ketoacyl-CoA thiolase deficiency The first isolated mammalian PEX gene is PEX2, which was first called peroxisome assembly factor-1 (PAF1).22 This gene 5. Dihydroxyacetone phosphate (DHAP) acyltransferase deficiency was cloned by means of functional complementation cloning 6. Alkyl-DHAP synthase deficiency using peroxisome-deficient Chinese hamster ovary (CHO) 7. Refsum disease (phytanoyl-CoA hydroxylase deficiency) cells. Expression library was transfected into these CHO mu- 8. Alpha-methylacyl-CoA racemase deficiency tants, cells with restored peroxisome biogenesis were selected, and the cDNA was recovered from these cells. Pex2p is a 35- 9. Acatalasemia kDa peroxisomal membrane protein and has two membrane 10. Hyperoxaluria type I (alanine glyoxylate aminotransferase deficiency) spanning regions and a RING finger motif. In 1992, we clari- 11. Mavalonate kinase deficiency fied that PEX2 restored biogenesis of peroxisomes in fibro- 12. Glutaric aciduria type 3 (glutaryl-CoA oxidase deficiency) blasts from a ZS patient who belonged to complementation group F.23 This patient carried a homozygous nonsense muta- tion R119Ter. A frameshift mutation del 550C, which destroys the second membrane spanning region, is associated with se- vere ZS, whereas another frameshift mutation del 642G, which destroys only the RING finger motif, is found in the milder IRD phenotype.24 A missense mutation E55K, which was iden- tified in a IRD patient, proved to be a ts mutation.18 The second gene identified in PBD patients is PEX5.25 This gene was cloned by expressed sequence tag (EST)-homology search using a hu- man database and information on the yeast PEX gene. Pex5p is a cytosolic receptor for peroxisomal targeting signal (PTS)-1. A Zellweger patient who belonged to Group 2 had a nonsense mutation R390Ter, and an NALD patient had a missense mu- tation. The PTS-1 receptor binds tightly to the PTS-1 motif of acyl-CoA oxidase, Ser-Lys-Leu (SKL), whereas the binding po- tential to Ala-Lys-Leu (AKL) in D-bifunctional protein or Lys- Ala-Asn-Leu (KANL) in catalase is weaker.26 PEX6, the gene Fig. 1 Electron micrograph and immunofluorescence staining of peroxisomes. A: Con- for group C PBD, was also cloned using the functional comple- trol hepatocytes (electron micrograph with diaminobenzidine staining). B: Control fibro- mentation method.27 Pex6p belongs to the group of AAA-pro- blasts (immunofluorescence staining with anticatalase). C: Zellweger hepatocytes 28 (electron micrograph with diaminobenzidine staining). D: Zellweger fibroblasts teins and is considered to interact with Pex1p. A splice mu- (immunofluorescence staining with anticatalase). tation and a one base insertion were identified in ZS patients.29 PEX12 encodes a peroxisomal