Molecular and genetic characterization of peroxisome biogenesis disorders Ebberink, M.S.

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1 UvA-DARE (Digital Academic Repository) Molecular and genetic characterization of peroxisome biogenesis disorders Ebberink, M.S. Link to publication Citation for published version (APA): Ebberink, M. S. (2010). Molecular and genetic characterization of peroxisome biogenesis disorders General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 28 Dec 2018

2 Molecular and genetic Hier komt characterization de tekst voor of de peroxisome rug; hoe dikker biogenesis de rug, hoe disorders groter de tekst Merel S. Ebberink Molecular and genetic characterization of peroxisome biogenesis disorders Merel S. Ebberink

3 Molecular and genetic characterization of peroxisome biogenesis disorders

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5 Molecular and genetic characterization of peroxisome biogenesis disorders Academisch proefschrift ter verkrijging van de graad van doctor aan d e Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op dinsdag 21 september 2010, te 14:00 uur door Merel Sanne Ebberink geboren te Utrecht

6 Promotiecommissie Promotor: Copromotoren: Overige leden: Prof. dr. R.J.A. Wanders Dr. H.R. Waterham Dr. S. Ferdinandusse Prof. dr. R.C.M. Hennekam Prof. dr. C.J.F. van Noorden Prof. dr. F.A. Wijburg Prof. dr. H.S.A. Heymans Dr. M. Fransen Dr. S.M. Houten Faculteit der Geneeskunde The work described in this thesis was carried out at the laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, The Netherlands. The research was financially supported by a grant of the Prinses Beatrix Fonds (MAR ) and by the FP6 European Union Project Peroxisomes (LSHG-CT ).

7 Table of contents Abbreviations 6 Chapter 1 Introduction 7 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Genetic classification and mutational spectrum of more than 600 patients with a Zellweger syndrome spectrum disorder Submitted for publication Genotype-phenotype correlation in PEX5-deficient peroxisome biogenesis defective cell lines Human Mutation (2009) 30(1):93-98 Spectrum of PEX6 mutations in Zellweger syndrome spectrum patients Human Mutation (2010) 31(1):E Identification of an unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene Journal of Medical Genetics (2010) Mutations in PEX10 are a cause of autosomal recessive ataxia Annals of Neurology (2010) A novel human peroxisome biogenesis disorder affecting peroxisome division In preparation for submission Summary and conclusions 115 Samenvatting 121 Dankwoord 125

8 Abbreviations AAA ATPase associated with diverse cellular activities mpts membrane peroxisomal targeting signal ACOX I acyl-coa oxidase I MR magnetic resonance AGT alanine-glyoxylate aminotransferase MRI magnetic resonance imaging ALDP adrenoleukodystrophy protein mrna messenger RNA AMACR 2-methylacylCoA racemase MRS magnetic resonance spectroscopy ATP adenosine triphosphate NALD neonatal adrenoleukodystrophy cdna complementary DNA NMDA N-methyl-D-aspartic acid CHO Chinese hamster ovary PAGE polyacrylamide gel electrophoresis DBP D-bifunctional protein PBD peroxisome biogenesis disorders DHA docasahexaenoic acid PCR polymerase chain reaction DHAP dihydroxyacetonephosphate PEG polyethylene glycol DHAPAT dihydroxyacetonephosphate acyltransferase PEX peroxin DHCA dihydroxycholestanoic acid PH1 primary hyperoxaluria type 1 DLP1 dynamin-like protein 1 PMP peroxisomal membrane protein DMEM Dulbecco s Modified Eagle Medium PTS peroxisomal targeting sequence DNA deoxyribonucleic acid RCDP rhizomelic chondrodysplasia punctata DRP dynamin-related protein RING really interesting new gene E1 ubiquitin activating enzyme RNA ribonucleic acid E2 ubiquitin-conjugating enzyme RT-PCR reverse transcriptase-pcr E3 ubiquitin-protein ligase SARA scale for assessment and rating of ataxia egfp enhanced green fluorescent protein SCPx sterol carrier protein X EMG electromyography SDS sodiumdodecylsulfate ENMG electroneuromyography SH3 scr homology 3 ER endoplasmic reticulum shrna short hairpin RNA ERG electroretinogram SKL peroxisomal targeting signal: Ser-Lys-Leu EST expressed sequence tags SNP single nucleotide polymorphism FCS fetal calf serum THCA trihydroxycholestanoic acid gdna genomic DNA TMD transmembrane domain GFP green fluorescent protein TPR tetrapeptide repeat HAM Ham s tissue culture medium Ubc ubiquitin-conjucating enzyme HEPES N-[2-Morpholino]ethanesulfonic acid VEP visual evoked potential hfis1 human fission 1 VLCFA very long chain fatty acid HRM high-resolution melting Vps1 vacuolar protein sorting-associated prot1 IF immunofluorescence X-ALD X-linked adrenoleukodystrophy IRD infantile refsum disease ZS Zellweger syndrome Mff mitochondrial fission factor ZSS Zellweger syndrome spectrum

9 Chapter 1Introduction

10 Chapter 1 Introduction 1 Peroxisomes are ubiquitous, single membrane bound organelles found in almost all eukaryotes, ranging from microorganisms (except archaezoa) to plants and animals. They contain enzymes for various metabolic pathways and protect the cell from toxic peroxides. Defects in peroxisomal functioning are the cause of a number of metabolic disorders with a spectrum of disease severities. Peroxisomes Peroxisomes were first described by Rhodin (Rhodin J, 1954). He discovered that mouse kidney cells contain small, apparently distinct organelles which he called microbodies. In 1966, De Duve and Baudhuin were the first to isolate these microbodies by differential and density gradient centrifugation (De Duve C. and Baudhuin, 1966). They discovered the presence of catalase and hydrogen peroxide (H 2 O 2 ) generating enzymes in these organelles, and for this reason they called them peroxisomes. Other members of the microbody family are glyoxysomes and glycosomes. Glyoxysomes were discovered in the seeds of germinating plants and named after the glyoxylate cycle (Breidenbach and Beevers, 1967). Glycosomes are found in kinetoplastids and contain enzymes for glycolysis (Opperdoes et al., 1977). To date, peroxisomes have been identified in every human cell, except in red blood cells. Typically, a human cell contains several hundred peroxisomes, and they are most abundant in liver and kidney. They are usually round or oval structures with a diameter of µm, but they can elongate to tubular structures. Peroxisomes in cultured mammalian cells have a half-life of approximately 2 days (Huybrechts et al., 2009). Functions of peroxisomes First insights into peroxisomal functions were gained when Brown et al reported elevated levels of very long chain fatty acids in plasma from patients with Zellweger syndrome (ZS) (Brown et al., 1982) and Heymans et al reported a deficiency of plasmalogens in erythrocytes and tissues from ZS patients (Heymans et al., 1983). Currently, it is known that human peroxisomes play an important role in various metabolic pathways, among which the α- and β-oxidation of fatty acids, the biosynthesis of ether-phospholipids, bile acids and poly-unsaturated fatty acids, and the degradation of purines, polyamines, L-pipecolic acid and D-amino acids (van den Bosch et al., 1992; Wanders and Waterham, 2006a). Below I will briefly describe the main peroxisomal metabolic pathways in humans. Fatty acid α-oxidation Fatty acids with a methyl-group at the third position, like phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), are not a substrate for β-oxidation. These fatty acids have to be shortened by one carbon atom via α-oxidation to produce the corresponding fatty acid with the methyl group at the second position (Casteels et al., 2003; Wanders et al., 2003). Fatty acid α-oxidation only takes place in the peroxisome and accepts acyl-coa esters as substrate. For this reason, phytanic 8

11 Introduction acid is first activated to the acyl-coa ester, i.e. phytanoyl-coa, and thereafter broken down by α-oxidation to pristanic acid, a suitable substrate for β-oxidation. Fatty acid β-oxidation Fatty acids are degraded from the carboxyl end in a cyclic process called β-oxidation. Each cycle of β-oxidation occurs via four steps; including dehydrogenation, hydration, dehydrogenation again, and thiolytic cleavage (Wanders et al., 2001a). In each cycle, the fatty acid carbon chain is shortened by 2 carbon atoms (or 3 carbon atoms in case of 2-methyl-branched-chain substrates). The peroxisomal β-oxidation system can only shorten fatty acids but cannot degrade the fatty acids fully into acetyl-coa units. The shortened fatty acids are exported from the peroxisomes and transferred to the mitochondria to be degraded to acetyl-coa, a substrate of the mitochondrial tricarboxylic acid cycle to generate a form of usable energy (ATP) for cell processes. Substrates for the peroxisomal β-oxidation are very long chain fatty acids (VLCFAs; C24:0), branched chain fatty acids such as pristanic acid, the bile acids intermediates di- and trihydroxycholestanoic acid (DHCA and THCA), long chain dicarboxylic acids (products of the ω-oxidation), and the side chains of eicosanoids. Furthermore, the peroxisomal β-oxidation system is involved in the last step of the biosynthesis of the poly-unsaturated fatty acid docosahexaenoic acid (DHA; Ferdinandusse et al., 2001). 1 Ether-phospholipid biosynthesis Ether-phospholipid biosynthesis starts in the peroxisome and is catalyzed by two peroxisomal membrane-bound proteins dihydroxyacetonephosphate acyltransferase (DHAPAT) and alkyl-dihydroxyacetonephosphate-synthase (alkyl-dhap-synthase). DHAPAT generates acyl-dhap from acyl-coa and dihydroxyacetonephosphate (DHAP), after which the ether bond is formed by the alkyl-dhap synthase resulting in alkyl-dhap. After conversion of alkyl-dhap into alkyl-g3p either in peroxisomes or the endoplasmic reticulum, the final steps of ether-phospholipid synthesis are performed by endoplasmic reticulum enzymes (Brites et al., 2004). Two groups of ether-phospholipids can be distinguished containing either an ether bond or a vinyl-ether bond. Ether-phospholipids with a vinyl ether bond at the sn-1 position are called plasmalogens. Plasmalogens are components of cellular membranes and are found in brain myelin, heart muscle, kidney, skeletal muscle, spleen, blood cells and in low levels in liver. Plasmalogens appear to have an antioxidant effect towards a wide variety of reactive species including, amongst others, reactive oxygen species (Zoeller et al., 1999) and iron-induced peroxidation (Sindelar et al., 1999). Other peroxisomal functions Other metabolic processes that occur in peroxisomes, at least partially, are the degradation of purines and pyrimidines, glyoxylate detoxification, L-pipecolic acid and D-amino acids degradation, hydrogen peroxide degradation, polyamide oxidation and fatty acid chain elongation. Because these metabolic processes are not studied in this thesis, they will not be described in detail here (for a recent review see; Wanders and Waterham, 2006a). 9

12 Chapter 1 1 Proteins involved in the biogenesis of peroxisomes Peroxins (PEX) are encoded by PEX genes and are proteins that play a role in peroxisomal protein import, peroxisome proliferation or peroxisome inheritance (collectively also called peroxisome biogenesis). Most of the PEX genes have been identified in one of yeast species using negative genetic screens, in which potential mutants were selected for their inability to grow on peroxisome-requiring carbon sources (Erdmann et al., 1989; Gould et al., 1992; Liu et al., 1992), or using positive screens that are based on H 2 O 2 -lethality, or the bleomycin import selection procedure in S. cerevisiae (Elgersma et al., 1993; Van der Leij et al., 1992). Human orthologues were often identified by similarity searching in the human expressed sequence tags (EST) database using the yeast PEX genes as query. A second PEX gene identification strategy used functional complementation of peroxisomedeficient Chinese hamster ovary (CHO) cells with mammalian cdna expression libraries (Gould and Valle, 2000). To date, 31 different peroxins have been identified in yeast that are involved in different peroxisomal processes, including the formation of peroxisomal membranes, peroxisomal growth, fission and proliferation, import of peroxisomal matrix proteins or peroxisome inheritance, whereas in humans, so far only 16 peroxins have been identified that are required for these processes. In humans, the following 16 peroxins have been identified: PEX5 and PEX7, which function as cytosolic receptors for peroxisomal matrix proteins; PEX13 and PEX14, which act as a docking site for the receptor-cargo complex; PEX3, PEX16 and PEX19, which are involved in the peroxisomal membrane assembly; PEX1, PEX6 and PEX26, which play a role in the recycling of the cytosolic receptors PEX5 and PEX7; PEX2, PEX10 and PEX12, which are involved in the ubiquitination of PEX5; and PEX11α, PEX11β, PEX11γ, which are involved in peroxisome proliferation. The role of each PEX is described in more detail in the following paragraphs. Peroxisome Biogenesis The biogenesis of peroxisomes is a multi-step process. First the peroxisomal membrane is synthesized consisting of lipids and proteins. The lipids first form a membrane and then the peroxisomal membrane proteins (PMPs) need to be targeted to and inserted into the lipid layer. Finally, the peroxisomal matrix proteins need to be imported to make metabolically functional peroxisomes. Besides the de novo formation of peroxisomes, another aspect of peroxisome biogenesis is the proliferation and division of peroxisomes. Formation of peroxisomes Originally, two models of peroxisome multiplication had been described in literature. In the first model, peroxisomes form de novo by vesicle budding from the endoplasmic reticulum (ER; Tabak et al., 2003; Tabak et al., 2006; Titorenko et al., 2000). This model was mainly based on electron microscopical observations, showing that peroxisomes were found in close proximity to the ER. In the second model, peroxisomes multiply by growth and division (Lazarow, 2003; Lazarow and Fujiki, 1985). This model was supported by the discovery that peroxisomal proteins are synthesized on free ribosomes and targeted posttranslationally to peroxisomes. 10

13 Introduction de novo Growth and fission Pre-peroxisome ER Import PMP Import matrix protein fission PEX11 PEX11 FIS1 DLP1 Mff 1 Figure 1. Model for peroxisome biogenesis and division. Peroxisomes grow from pre-peroxisomal vesicles originating from specialized compartments of the endoplasmic reticulum (ER), which can develop to a metabolically active peroxisome or, through the direct import of matrix and membrane proteins, in mature peroxisomes. The division of peroxisomes proceeds through three steps: elongation of peroxisomes, membrane constriction, and finally, fission of peroxisomes. The PEX11 proteins are implicated in the elongation and constriction step, whereas DLP1 and FIS1 catalyze the fission step. More recent data strongly suggest that peroxisome biogenesis in mammalian cells occurs predominantly by fission of pre-existing peroxisomes, but that peroxisomes can be formed also de novo (e.g. when lacking peroxisomes), either ER-dependent or independent (Figure 1). This view is based on several observations. For example, human fibroblast cells with a defect in PEX3, PEX16 or PEX19 lack peroxisomal remnants, but genetic complementation restores peroxisome biogenesis (South and Gould, 1999). In addition, in temperature-sensitive mutants of H. polymorpha it was shown that peroxisome biogenesis can be restored in cells lacking peroxisomes (Waterham et al., 1993). Moreover, based on live cell imaging approaches Kim et al. concluded, that the increase in peroxisome number in growing mammalian wild-type cells results primarily from new peroxisomes derived from the ER rather than by division of pre-existing peroxisomes (Kim et al., 2006). However, different proteins of the peroxisomal division machinery have been identified in yeast, mammalian and plant cells, which support the growth and division model (Delille et al., 2009). In addition, a recent study on peroxisome dynamics in cultured mammalian cells revealed that the matrix content of pre-existing peroxisomes is not evenly distributed over new peroxisomes. The authors of this study propose two hypotheses: (1) new peroxisomes are formed de novo from the ER or another pre-peroxisomal template or (2) peroxisomes generally multiply by growth and non-symmetrical fission events (Huybrechts et al., 2009). Division of peroxisomes Different proteins have been identified that are involved in the division of peroxisomes. The division of peroxisomes consists of elongation of the peroxisome, membrane constriction and finally fission of peroxisomal tubules (Fagarasanu et al., 2007; Hettema and Motley, 2009; Kaur and Hu, 2009; Figure 1). The following proteins are involved in the division of peroxisomes (and mitochondria): (1) Dynamin-like 11

14 Chapter 1 1 protein 1 (DLP1, in mammals) and vacuolar protein sorting-associated protein 1 (Vps1, in yeast). Cells lacking DLP1 have elongated peroxisomal tubules which are constricted, but cannot divide, whereas, the mitochondria are elongated, entangled, tubular structures (Koch et al., 2004; Waterham et al., 2007). (2) Mammalian Fission 1 (hfis1), a tail-anchored protein of the outer mitochondrial membrane (Kobayashi et al., 2007; Koch et al., 2005). An increase of hfis1 promotes mitochondrial and peroxisomal division, while the loss of hfis1 inhibits the division of both organelles. hfis1 acts as a DLP1 receptor anchored at the mitochondrial outer membrane (Serasinghe and Yoon, 2008) and has probably the same function in peroxisomes. (3) Mammalian mitochondrial fission factor (Mff), a tail-anchored protein. The loss of Mff results in the elongation of both organelles (Gandre-Babbe and van der Bliek, 2008). (4) Members of the PEX11 family. These membrane proteins can induce peroxisome proliferation by elongation of the organelle in mammalian, yeast and plant cells (Erdmann and Blobel, 1995; Lingard et al., 2008; Marshall et al., 1995). In mammals, three PEX11 isoforms (PEX11α, PEX11β and PEX11γ) have been identified. They are all integral peroxisomal membrane proteins (PMPs) and have both their aminoand carboxy-terminal ends exposed to the cytosol (Abe and Fujiki, 1998; Schrader et al., 1998). PEX11β interacts with itself and in addition interacts with Fis1 by direct binding to its C-terminal region (Kobayashi et al., 2007). Overexpression of PEX11α and PEX11β results in peroxisome proliferation. In contrast, PEX11γ overexpression has no effect on peroxisome abundance (Li et al., 2002b). PEX11β knockout mice displayed a twofold reduction in peroxisome abundance (Li et al., 2002a). We recently identified the first patient with a defect in PEX11β, who displayed a defect in peroxisome division. Fibroblasts of this patient revealed an import deficiency in 5-10% of the peroxisomes. Furthermore, the peroxisomes were elongated and arranged in rows compared to control fibroblasts (see Chapter 7). Peroxisomal membrane protein import The peroxisomal membrane is composed of phospholipids, mainly phosphatidyl choline and phosphatidyl ethanolamine (Schneiter et al., 1999), and PMPs. The phospholipids are synthesized in the ER, but it is unknown how they are transported to the peroxisome. PMPs are synthesized on free cytosolic ribosomes and targeted posttranslationally to the peroxisome. Two targeting routes for PMPs have been identified: some are targeted from the cytoplasm directly to the peroxisomal membrane; others are sorted indirectly to peroxisomes by way of ER-derived vesicles or a specialized subdomain of the ER (Van Ael and Fransen, 2006). The targeting and insertion of PMPs into the peroxisomal membrane is probably mediated by a membrane peroxisomal targeting signal (mpts). Unlike the signal responsible for the targeting of peroxisomal matrix proteins (described below), mptss do not contain a recognisable consensus. Instead, several features within the PMP are important for its targeting. For instance, in several PMPs a well conserved short stretch of positively charged residues and hydrophobic membrane-spanning domains have been identified. Disruption of conserved residues in these sites disturbed proper targeting of PMP34 (Honsho and Fujiki, 2001) and the peroxisomal membrane protein PEX13 (Rottensteiner et al., 2004). Several groups have reported that PMPs contain more than one targeting signal (Iwashita et al., 2009; Jones et al., 2001; Wang et al., 2001; Wang et al., 2004). Although no universal mpts consensus 12

15 Introduction sequence has been identified, the mptss consist of at least two functionally distinct domains: (1) a targeting element, and (2) an adjacent hydrophobic segment, which is required for the permanent insertion of the protein into the peroxisomal membrane (Rottensteiner et al., 2004; Van Ael and Fransen, 2006). PEX3, PEX16 and PEX19, have been implicated to play a role in the targeting and insertion of PMPs into the peroxisomal membrane. The essential role of these PEXs in the membrane import system was first concluded from the absence of peroxisomal remnants (ghosts) in patient cell lines lacking one of these proteins (Honsho et al., 1998a; Matsuzono et al., 1999; Muntau et al., 2000; Shimozawa et al., 2000). 1 It has been suggested that PEX19 is either a chaperone for newly synthesized PMPs in the cytosol or functions as a PMP import receptor. PEX19 is a farnesylated protein and has a high affinity for a broad range of PMPs (Iwashita et al., 2009; Matsuzono et al., 2006; Sacksteder and Gould, 2000; Snyder et al., 2000). PEX19 is predominantly localized in the cytosol; however, a small portion of PEX19 is associated with the peroxisomal membrane. Disruption of conserved residues in binding sites of PEX19 for PMPs resulted in mislocalization of the respective proteins (Jones et al., 2004). In contrast, other studies reported that PEX19-PMP interaction takes place in regions distinct from those involved in targeting (Fransen et al., 2001). PEX19 seems to be essential for the targeting of many, but not all, PMPs to the peroxisomal membrane. It has been suggested that two independent mpts pathways exist: one depending on PEX19 (class I PMPs) and a second, independent of PEX19 (class II PMPs). PEX3 would be a class II PMP, because PEX19 has no effect on the import of PEX3 into pre-existing peroxisomes (Fujiki et al., 2006; Jones et al., 2004). PEX3 interacts with PEX19, but this binding is not to the mpts of PEX3 (Fransen et al., 2004; Sacksteder et al., 2000). In the current model, PEX3 functions as the docking factor for PEX19 on the peroxisomal membrane (Fang et al., 2004; Fransen et al., 2005; Pinto et al., 2009). The PEX19-PMP complex interacts with PEX3, and this interaction results in the insertion of the PMP into the peroxisomal membrane (Pinto et al., 2006). PEX3 is a membrane protein possessing one transmembrane domain near its N-terminus and exposing its C-terminus to the cytosol (Ghaedi et al., 2000; Halbach et al., 2009; Soukupova et al., 1999). Pinto et al hypothesized that the cytosolic portion of PEX3, the domain that binds PEX19-PMP complexes, interacts with membrane lipids to perturb the peroxisomal lipid bilayer to allow the PMP to insert in the peroxisomal membrane (Pinto et al., 2009). Recently, results have been published indicating an additional function for PEX3. S. cerevisiae PEX3 functions as an anchor for Inp1 at the peroxisomal membrane, which is required for peroxisome segregation (Munck et al., 2009). PEX16 has two putative membrane-spanning domains and exposes its C- and N-terminal domains to the cytosol (Honsho et al., 2002; South and Gould, 1999). The role of PEX16 in the import of PMPs into the peroxisomal membrane is unclear. Two different groups of PEX16-defective patients have been reported; patients with a severe clinical presentation of which the fibroblasts displayed a defect in import of peroxisomal matrix and membrane proteins, resulting in a total absence 13

16 Chapter Membrane protein Figure 2. Model for peroxisomal membrane protein import. PEX19 functions as a cycling chaperone/ receptor for PMPs. In the cytosol PEX19 binds PMPs and delivers them to the peroxisomal membrane. The PEX19-PMP complex docks on the peroxisomal membrane via interaction with PEX3. of peroxisomal remnants (Honsho et al., 1998b; Shimozawa et al., 2002) and, very recently, a number of patients with a relatively mild clinical phenotype of whom the fibroblasts showed enlarged, protein import-competent peroxisomes (Ebberink et al, 2010 and chapter 5). Recent data indicate that PEX16 functions as a docking site for PEX3 and serves as the peroxisomal membrane receptor for PEX3-PEX19 complexes (Matsuzaki and Fujiki, 2008). In Y. lipolytica, PEX16 is involved in peroxisome proliferation and has no role in membrane assembly (Eitzen et al., 1997). Figure 2 shows a model for the import of PMPs. PEX19 binds newly synthesized PMPs in the cytosol. The PEX19-PMP complex docks by binding to PEX3 at the peroxisomal membrane. PMPs are inserted into the peroxisomal membrane and PEX19 recycles back to the cytosol. Peroxisomal matrix protein import The targeting of peroxisomal matrix proteins to peroxisomes is mediated by peroxisomal targeting sequences (PTS), which are recognized by specific cytosolic receptor proteins. Two cytosolic receptors have been identified that can recognize and transport PTS1- and PTS2- containing peroxisomal matrix proteins to the peroxisome. These are PEX5 and PEX7 respectively. The majority of peroxisomal proteins contains a carboxyl-terminal tripeptide with the following conserved consensus sequence, [S/A/C][K/R/H][L/M] (Elgersma et al., 1996), called PTS1, which is recognized by the cytosolic receptor protein PEX5. In addition, a few proteins have an amino-terminal PTS2 with the consensus sequence [R/K][L/V]X5[H/Q][L/A] (Swinkels et al., 1991), which is recognized by the cytosolic receptor protein PEX7 (Dodt et al., 2001a). A number of proteins are imported into the peroxisome that lack a recognisable PTS1 or PTS2 consensus. In the yeast S. cerevisiae, acyl-coa oxidase is imported by PEX5 independent of the PTS1 signal (Klein et al., 2002). 14

17 Introduction Several groups have suggested that peroxisomal matrix proteins lacking a PTS1 and PTS2 signal are imported via piggy backing ; matrix proteins form hetero-oligomeric complexes in the cytosol with at least one protein containing a PTS1 signal. These complexes are then recognized by PEX5, which transports and imports the whole complex into the peroxisome. The phenomenon piggy backing has been shown in yeast only with artificial substrates (Glover et al., 1994; Yang et al., 2001). In human fibroblasts, it has been shown that colloidal gold particles conjugated to biotinylated human serum albumin-skl can be translocated into the peroxisomal matrix (Walton et al., 1995). 1 The peroxisomal receptors PEX5 and PEX7 Human PEX5 is a 67-kD protein with seven di-aromatic pentapeptide repeats (WxxxF/Y) in its amino-terminal half and seven tetrapeptide repeats (TPRs) in its carboxy-terminal half (Gatto et al., 2000). The TPR-containing carboxy-terminal half of PEX5 has been shown to mediate the interaction with the PTS1 sequence, whereas the WxxxF/Y motifs in the amino-terminal half of PEX5 appear essential for docking to the peroxisomal membrane and for binding to either PEX13 or PEX14 (Saidowsky et al., 2001; Weller et al., 2003). Previous studies established that the PEX5 amino acid residues are involved in PTS1 recognition and that the PTS1-binding domain in PEX5 is formed by the two carboxy-terminal TPR motif clusters TPR1-3 and TPR5-7 and the 7C-loop. The two TPR motif clusters are 5 A 5 5 D B 5 Ub PTS1 protein C Figure 3. A model for peroxisomal matrix protein import. Peroxisomal protein import can be divided into four stages: A) Binding of peroxisomal matrix proteins to their receptor. B) Docking of the receptor-cargo complex to the peroxisome. C) Translocation of the receptor-cargo complex and release of the cargo into the matrix. D) Receptor recycling into the cytosol or receptor degradation via the proteosome. The PEX proteins are depicted by their numbers. 15

18 Chapter 1 1 thought to surround the PTS1 protein almost completely, while TPR4 forms a distinct hinge structure (Gatto et al., 2000; Stanley et al., 2006; Stanley et al., 2007). The 7C-loop is required to connect the two TPR clusters. The pentapeptide repeats 2-4 are required for binding PEX13, and the amino-terminal pentapeptide repeats are required for binding PEX14 (Otera et al., 2002; Saidowsky et al., 2001) In humans, two functional protein variants of PEX5 are produced as a result of alternative splicing of the PEX5 mrna. The longest variant, PEX5L, contains an additional 111bp encoding 37 amino acids, due to alternative splicing of exon 7 (Dodt et al., 2001a). The shorter protein, PEX5S, has been reported to be exclusively involved in peroxisomal PTS1 protein import, whereas PEX5L mediates both PTS1 and PTS2 protein import. In fact, docking of the PEX7-PTS2 protein complex to the PEX13 and PEX14 proteins of the peroxisomal import machinery can only occur through physical interaction with PEX5L. The PEX5L region involved in this interaction, amino acid residues , includes part of the PEX5L-specific insertion (Braverman et al., 1998; Dodt et al., 2001a; Matsumura et al., 2000). PEX7, the PTS2 receptor, contains six tryptophan aspartate (WD) repeats, which are each approximately 40 amino acids long and contain a central WD motif. PEX7 requires additional proteins for the import of PTS2 proteins; PEX18 or PEX21 in S. cerevisiae (Purdue et al., 1998), PEX20 in Y. lipolytica (Einwachter et al., 2001) and Neurospora crassa (Sichting et al., 2003), and PEX5L in mammals (Dodt et al., 2001b; Otera et al., 2000). In line with the above described functions, defects in PEX5 in principle result in an inability to import PTS1 and PTS2 proteins leading to a generalized peroxisome biogenesis defect (Dodt et al., 1995; Ebberink et al., 2009; Chapter 3) whereas a defect in PEX7 only affects the import of a small subset of peroxisomal proteins leading to a different clinical presentation, i.e. Rhizomelic Chondrodysplasia Punctata (RCDP; Motley et al., 2002a). Receptor docking and release of cargo The current model for PEX5-mediated PTS1 protein import can be divided into four stages (Figure 3): 1) PEX5 binds to newly synthesized PTS1 proteins in the cytosol and transports them to the peroxisomal membrane. 2) Docking of the receptorcargo complex to the peroxisome. 3) Release of the cargo into the peroxisomal matrix. 4) Receptor recycling into the cytosol for another round of import or receptor degradation via the proteasome (Platta et al., 2007). After the receptors PEX5 and PEX7 have bound their cargo, the receptor-cargo complex is transported to the peroxisomal membrane. The transmembrane proteins PEX13 and PEX14 have been implicated in the docking step. PEX13 contains 2 transmembrane domains as well as an Src homology 3 (SH3) domain (Gould et al., 1996). PEX13 interacts with PEX14 and both the PEX5 and PEX7 receptors. PEX14 is an integral membrane protein which interacts with itself, PEX13, PEX5, PEX7 and PEX19. It is believed that PEX14 represents the initial docking site for both receptor proteins, because PEX14 has a higher affinity for cargo-loaded PEX5, whereas PEX13 has a higher affinity for PEX5 alone (without cargo; Urquhart et al., 2000). Neither PEX13 nor PEX14 have the capacity to bind peroxisomal matrix proteins suggesting that PEX5 does not just deliver its cargos to these transmembrane 16

19 Introduction peroxins (Gould and Collins, 2002). After docking of the receptor-cargo complex to the peroxisomal membrane, the peroxisomal matrix protein needs to be translocated into the peroxisomal matrix and the receptor needs to be recycled to the cytosol. It is uncertain if PEX5 inserts into the peroxisomal membrane or even enters completely the peroxisome to release the cargo (Dammai and Subramani, 2001). Studies with mammalian PEX5 suggest that PEX5 inserts into the peroxisomal membrane, exposing a small N-terminal domain to the cytosol and another part to the peroxisomal matrix (Gouveia et al., 2000; Gouveia et al., 2003). 1 Recycling of the receptors The receptors are recycled to the cytosol after they have released the cargo into the peroxisomal matrix. Like in yeast, the mammalian PEX5 is ubiquitinated, either mono-ubiquitinated, which is a sign for recycling, or poly-ubiquitinated, which makes PEX5 substrate for the proteasome for degradation (Carvalho et al., 2007). Different peroxins have been implicated in these steps. Ubiquitination is the covalent linkage of the 8-kDa protein ubiquitin to a lysine residue in a protein and occurs in three steps; Ubiquitin is activated in an ATPdependent manner by an ubiquitin activating enzyme (E1), then it is transferred to an ubiquitin-conjugating enzyme (Ubc, E2), which attaches Ub to the target protein with support of an ubiquitin-protein ligase (E3). The RING zinc finger proteins PEX2, PEX10 and PEX12 belong to the ubiquitin-protein ligases family; they are all integral peroxisomal membrane proteins and have a cytosolic carboxy-terminal zinc-binding domain. Cells lacking one of these zinc-binding proteins accumulate PEX5 at the peroxisomal membrane, which suggests that these proteins act downstream of the receptor docking (Chang et al., 1999; Dodt et al., 2001b; Dodt and Gould, 1996). PEX10 and PEX12 interact with each other and both proteins can also directly bind the PTS1 receptor PEX5. Different studies investigated the role of PEX2, PEX10 and PEX12 in the ubiquitination of PEX5. Platta et al report that in S. cerevisiae PEX2 is involved in the Ubc4-dependent poly-ubiquitination of PEX5 and that PEX12 is involved in the PEX4 dependent mono-ubiquitination of PEX5 (Dodt and Gould, 1996; Platta et al., 2009). In contrast, Williams et al have shown that PEX10 acts as the E3 ligase for Ubc4p-dependent ubiquitination of PEX5 but not PEX4-dependent ubiquitination (Dodt and Gould, 1996; Williams et al., 2008). In addition, yeast PEX10 was shown to interact with PEX4 in vivo (Eckert and Johnsson, 2003). Yeast PEX4, is a member of the ubiquitin-conjugating enzyme family and is involved in the mono ubiquitination of PEX5. PEX4 is a peripherally associated PMP, located at the cytosolic face of the peroxisome. The protein is kept at the peroxisomal membrane via interaction with PEX22, an integral PMP (Dodt and Gould, 1996; Koller et al., 1999). Yeast PEX4 appears to have 3 human cytosolic ortholoques E2D1, E2D2, and E2D3 (UBcH5a/b/c; Grou et al., 2008) and each protein can promote the ubiquitination of PEX5 on its own. PEX1 and PEX6 have been implicated in the recycling of the receptors, and are both members of the AAA protein family (ATPases associated with various cellular activities). These proteins contain highly conserved domains of 230 amino acids, 17

20 Chapter 1 1 which contain Walker ATP binding sequences and have ATPase activity (Dodt and Gould, 1996; Yahraus et al., 1996). Both proteins were found to interact with each other (Dodt and Gould, 1996; Faber et al., 1998). The transmembrane protein PEX26 was shown to anchor PEX6 and PEX1 to the peroxisomal membrane in a PEX6-dependent manner. PEX1 and PEX6 are both important for the stability of PEX5, and they function in the terminal steps of the matrix protein import system (Dodt and Gould, 1996; Yahraus et al., 1996). It is suggested that they are involved in the release of PEX5 from the peroxisomal membrane. The recycling of PEX5 to the cytosol is an ATP-dependent process (Oliveira et al., 2003); and PEX1 and PEX6 play a role in this process (Grou et al., 2009). Peroxisomal disorders Defects in peroxisomal functioning are the cause of a number of peroxisomal disorders. Peroxisomal disorders can be categorized in two main groups; the peroxisome biogenesis disorders (PBDs), which have a defect in the biogenesis of peroxisomes and the single peroxisomal enzyme deficiencies, in which a single peroxisomal protein is functionally deficient (Depreter et al., 2003; van den Bosch et al., 1992; Wanders and Waterham, 2006a; Weller et al., 2003). Peroxisome biogenesis disorders Peroxisome biogenesis disorders are autosomal recessive disorders and include the Zellweger syndrome spectrum (ZSS) disorders and Rhizomelic Chondrodysplasia Punctata type I (RCDP; Gould and Valle, 2000). The ZSS include three phenotypes; Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD), which represent a spectrum of disease severity with ZS being the most, and IRD the least severe disorder (Weller et al., 2003). Clinically, patients with ZS (or cerebro-hepato-renal syndrome) have typical facial dysmorphisms that include a high forehead, hypoplastic supraorbital ridges, epicanthal folds and a broad nasal bridge. At birth, ZS patients have often large fontanelles (Gould et al., 2001a; Weller et al., 2003). They have ocular abnormalities (cataracts, glaucoma, corneal clouding, pigmentary retinopathy and optic nerve dysplasia), often sensorineural deafness, and profound neurological abnormalities (Barth et al., 2001; Gould et al., 2001b). In addition, the patients are severely hypotonic and have neonatal seizures. The development of internal organs (brain, liver and kidney) and the skeleton is disturbed. ZS patients generally die within the first year of life. Patients with NALD suffer from neonatal hypotonia and seizures, like ZS patients. In addition, they may suffer from progressive white matter disease, and usually die in late infancy (Kelley et al., 1986; Wanders and Waterham, 2005). Patients with IRD have no neuronal migration defect, but can develop a progressive white matter defect. Their cognitive and motor development varies between severe global handicap and a moderate learning disorder with deafness and visual impairment due to retinopathy. Their survival is variable, but most patients survive beyond infancy and some even reach adulthood (Poll-The BT et al., 1987). ZSS disorders can be caused by a defect in any of at least 12 different PEX genes (Steinberg et al., 2006). ZSS disorders are characterized by the absence of functional 18

21 Introduction peroxisomes (Goldfischer et al., 1973). The absence of functional peroxisomes causes biochemical abnormalities in plasma, erythrocytes and fibroblasts of patients with a ZSS disorder (Table 1). The peroxisomal biochemical profile (see Table 1) can be used to distinguish the PBD (ZSS and RCDP type I) from the single enzyme defects in peroxisomal fatty acid metabolism. 1 RCDP type I is clinically characterized by unique skeletal abnormalities (mainly severe shortening and disturbed ossification of the proximal limbs and prominent calcific stippling at the knee, hip, elbow, and shoulder, and the vertebral clefts in vertebrae), cataracts, periarticular calcifications, multiple joint contractures, and mental retardation (Weller et al., 2003). Most RCDP patients die in the first months of life. Patients with RCDP type I have mutations in the PEX7 gene encoding peroxin 7, the cytosolic PTS2-receptor protein. PEX7 is required for targeting of a subset of enzymes to peroxisomes. In cells of patients with RCDP type I, the enzymes with a PTS2 signal are mislocalized to the cytoplasm instead of to the peroxisomes (Braverman et al., 1997; Braverman et al., 2002; Motley et al., 2002b). The mislocalization of enzymes with a PTS2 signal causes a defect in the α-oxidation of phytanic acid and ether-phospholipid biosynthesis (Table 1). Table 1. Biochemical parameters in plasma, erythrocytes and cultured skin fibroblasts of patients with a peroxisome biogenesis disorders. Plasma Erythrocytes Fibroblasts PBD ZSS RCDP I β-oxidation: - VLCFA (C26:0) levels N - Pristanic acid levels N - Docosahexanoic acid (DHA) - Bile acid intermediates N α-oxidation: - Phytanic acid Pipecolic acid oxidase: - L-Pipecolic acid N Etherphospholipid biosynthesis: - Plasmalogen levels β-oxidation: - C26:0 oxidation N - Pristanic acid oxidation N α-oxidation: - Phytanic oxidation Etherphospholipid biosynthesis: - Plasmalogen synthesis Organellar integrity: - Peroxisomes absent, normal reduced number Gene PEX genes PEX7 N, normal; PBD, peroxisome biogenesis disorder; ZSS, Zellweger syndrome spectrum; RCDP I, Rhizomelic Chondrodysplasia Punctata type I; VLCFA, very long chain fatty acid PEX genes: PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, PEX26 19

22 Chapter 1 1 Single peroxisomal enzyme deficiencies Single enzyme deficiency disorders are caused by mutations in a gene encoding either a peroxisomal enzyme or peroxisomal membrane transporter protein. They can be subdivided into subgroups on the basis of the peroxisomal metabolic pathway affected (Wanders and Waterham, 2006b). Table 2 lists the biochemical profile of the single enzyme deficiencies. The following single enzyme deficiencies are known: - X-linked adrenoleukodystrophy (X-ALD) is the most frequent single enzyme deficiency. X-ALD include three phenotypes; the rapidly progressive childhood cerebral form (CCALD), the milder adult form adrenomyeloneuropathy (AMN), and variants without neurologic involvement. Patients with X-ALD accumulate VLCFAs in their plasma and tissue, and are clinically characterized by progressive white matter demyelination. A defect in the ABCD1 gene, encoding a transmembrane transporter protein, causes X-ALD (Kemp and Wanders, 2007; Moser et al., 2001). - Acyl-CoA oxidase (ACOX I) is involved in the β-oxidation of VLCFAs. Patients with an ACOX I deficiency accumulate VLCFAs in their plasma and fibroblasts (Ferdinandusse et al., 2007; Poll-The et al., 1988). - D-bifunctional protein (DBP) holds a central position in the peroxisomal fatty acid beta-oxidation pathway and is involved in the beta-oxidation of C26:0, pristanic acid, as well as the bile acid intermediates DHCA and THCA (Watkins et al., 1989). In general, patients with a DBP deficiency accumulate VLCFAs, pristanic acid, and DHCA and THCA in plasma and tissues (Ferdinandusse et al., 2006a). - 2-Methylacyl-CoA racemase (AMACR) plays a crucial role in the degradation of fatty acids with a methyl group in the (2R)-configuration. Patients with an AMACR deficiency accumulate pristanic acid, THCA and DHCA (Ferdinandusse et al., 2000). - Refsum disease is caused by a defect in phytanoyl-coa hydroxylase, the second enzyme of the α-oxidation system, which results in accumulation of phytanic acid (Jansen et al., 1997; Jansen et al., 2000; Wanders et al., 2001b). - Sterol carrier protein X (SCPx) is a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. So far, one patient has been described with SCPx deficiency, who showed normal oxidation of C26:0 whereas the beta-oxidation of pristanic acid was markedly deficient (Ferdinandusse et al., 2006b). - RCDP type II is caused by a defect in DHAPAT, the first enzyme of the ether phospholipid biosynthesis pathway. Patients with RCDP type II have decreased levels of ether phospholipids, including plasmalogens (Wanders et al., 1992). 20

23 Introduction - RCDP type III is caused by a defect in alkyl-dhap-synthase, the second enzyme of the ether phospholipid biosynthesis pathway. Patients with RCDP type III have decreased levels of ether phospholipids, including plasmalogens (Wanders et al., 1994). - Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder caused by a deficiency of alanine-glyoxylate aminotransferase (AGT), which is encoded by the AGXT gene. PH1 is characterized by excessive endogenous oxalate production, which leads to impaired renal function (Williams et al., 2009). 1 - Another single peroxisomal enzyme deficiency without severe clinical consequences is acatalasaemia due to a defect in peroxisomal catalase (Eaton and Mouchou, 1995). Table 2. Biochemical parameters of single enzyme deficiencies in plasma, erythorocytes and fibroblasts of patients with a peroxisomal single enzyme deficiency. Single Enzyme Deficiencies RCDP II RCDP III X-ALD ACOX I DBP SCPx AMACR Plasma β-oxidation: - VLCFA (C26:0) levels N N N N - Pristanic acid levels N N N N - Bile acid intermediates N N N N α-oxidation: - Phytanic acid N N N N Erythrocytes Etherphospholipid biosynthesis: - Plasmalogen levels N N N N N Fibroblasts Organellar integrity: - Peroxisomes normal normal normal enlarged enlarged normal normal Gene GNPAT AGPS ABCD1 ACOX1 HSD17B4 SCP2 AMACR Enzyme DHAPAT ADHAPS ALDP ACOX1 DBP SCPx AMACR RCDP II and III, Rhizomelic Chondrodysplasia Punctata type II and III; X-ALD, X-linked adrenoleukodystrophy; ACOX I, Acyl-CoA oxidase; DBP, D-bifunctional protein; SCPx, Sterol carrier protein X; AMACR, 2-Methylacyl-CoA racemase; VLCFA, very long chain fatty acid; GNPAT, glyceronephosphate O-acyltransferase; AGPS, alkylglycerone phosphate synthase; ABCD1, ATP-binding cassette, sub-family D member 1; HSD17B4, hydroxysteroid (17-beta) dehydrogenase 4; DHAPAT, dihydroxyacetonephosphate acyltransferase; ADHAPS, alkyl-dihydroxyacetonephosphate-synthase; ALDP, adrenoleukodystrophy protein 21

24 Chapter 1 1 Outline of this thesis The main goal of the project was the genetic classification of cells from patients diagnosed with a peroxisome biogenesis disorder, with the aim to identify new complementation groups of peroxisome biogenesis disorders (PBD). To accomplish this goal, more than 600 primary skin fibroblasts of diagnosed PBD patients of the laboratory collection had to be categorized into known and possibly novel genetic complementation groups. Previous cell fusion complementation studies using patient fibroblasts revealed the existence of 12 distinct genetic complementation groups, representing defects in 12 different PEX genes. Initially, the same cell fusion complementation assay was used (Brul et al., 1988), which consists of fusing a ZSS cell line by means of polyethylene glycol treatment with a series of tester cell lines each representing a known PEX complementation group. The resulting multinucleated cells are then examined by catalase immunofluorescence microscopy to assess genetic complementation, i.e. re-appearance of peroxisomes due to defects in different genes, vs. non-complementation, i.e. peroxisomes remain absent due to mutations in the same gene. Because this assay was rather laborious, we developed a more rapid and direct complementation assay taking advantage of the known identities of the 12 human PEX genes currently implicated in peroxisome biogenesis. In this assay, patient ZSS cells with an unknown PEX gene defect are co-transfected separately with the different PEX cdnas and a fluorescent peroxisomal reporter protein allowing immediate visual detection of peroxisomes when present. Chapter 2 describes the assignment of more than 600 patients with a ZSS disorder to the 12 different complementation groups followed by genetic analysis of most of the cell lines. In chapter 3 and 4, we report novel mutations found in the PEX5 and PEX6 genes, respectively. The PTS1- and PTS2- protein import capacity was assessed in the cell lines with a defect in PEX5. This revealed that the location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in these cell lines (Chapter 3). To identify the mutations in PEX6, we developed a post-pcr high-resolution melting (HRM) curve assay to scan the PEX6 gene for potential sequence variations (Chapter 4). Chapter 5 describes six patients with an unusual variant peroxisome biogenesis disorder. The patients display an unexpected mild variant peroxisome biogenesis disorder due to mutations in the PEX16 gene. Surprisingly, their fibroblasts showed enlarged, import-competent peroxisomes. In the last years, several patients with a milder form of a ZSS disorder have been diagnosed, which display different degrees of peroxisomal mosaicism, i.e. cells completely lacking functional peroxisomes mixed with cells containing functional peroxisomes. As previously described, such cell lines often lose the mosaicism after culturing at elevated temperature resulting in a nearly complete peroxisome deficiency (Gootjes et al., 2004). In addition, most of these cell lines show temperature sensitivity of biochemical parameters. Chapter 6 describes two patients with a mild form of a ZSS disorder, who display a peroxisomal mosaicism of approximately 80% cells with functional peroxisomes. The peroxisomal mosaicism was still observed at 40 C. The patients, a child and an adult, have a normal intelligence, progressive ataxia, axonal 22

25 Introduction motor neuropathy, decreased vibration sense and have marked cerebellar atrophy. The defect in the PEX10 gene could only be identified by sequencing of PEX genes. In chapter 7, we describe the identification of a novel peroxisomal disorder. The patient has a defect in PEX11β, causing a mild Zellweger syndrome spectrum disorder, with no peroxisomal biochemical abnormalities in plasma, erythrocytes and cultured fibroblasts, but with a defect of the fission of peroxisomes. 1 References Abe I, Fujiki Y cdna cloning and characterization of a constitutively expressed isoform of the human peroxin Pex11p. Biochem Biophys Res Commun 252: Barth PG, Gootjes J, Bode H, Vreken P, Majoie CB, Wanders RJ Late onset white matter disease in peroxisome biogenesis disorder. Neurology 57: Braverman N, Steel G, Obie C, Moser A, Moser H, Gould SJ, Valle D Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata. Nat Genet 15: Braverman N, Dodt G, Gould SJ, Valle D An isoform of pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum Mol Genet 7: Braverman N, Chen L, Lin P, Obie C, Steel G, Douglas P, Chakraborty PK, Clarke JT, Boneh A, Moser A, Moser H, Valle D Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype. Hum Mutat 20: Breidenbach RW, Beevers H Association of the glyoxylate cycle enzymes in a novel subcellular particle from castor bean endosperm. Biochem Biophys Res Commun 27: Brites P, Waterham HR, Wanders RJ Functions and biosynthesis of plasmalogens in health and disease. Biochim Biophys Acta 1636: Brown LM, Duck-Chong CG, Hensley WJ Improved procedure for lecithin/sphingomyelin ratio in amniotic fluid reduces false predictions of lung immaturity. Clin Chem 28: Brul S, Westerveld A, Strijland A, Wanders RJ, Schram AW, Heymans HS, Schutgens RB, van den Bosch H, Tager JM Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis. J Clin Invest 81: Carvalho AF, Pinto MP, Grou CP, Alencastre IS, Fransen M, Sa-Miranda C, Azevedo JE Ubiquitination of mammalian Pex5p, the peroxisomal import receptor. J Biol Chem 282: Casteels M, Foulon V, Mannaerts GP, Van Veldhoven PP Alpha-oxidation of 3-methyl-substituted fatty acids and its thiamine dependence. Eur J Biochem 270: Chang CC, Warren DS, Sacksteder KA, Gould SJ PEX12 interacts with PEX5 and PEX10 and acts downstream of receptor docking in peroxisomal matrix protein import. J Cell Biol 147: Dammai V, Subramani S The human peroxisomal targeting signal receptor, Pex5p, is translocated into the peroxisomal matrix and recycled to the cytosol. Cell 105: De Duve C., Baudhuin P Peroxisomes (microbodies and related particles). Physiol Rev 46: Delille HK, Alves R, Schrader M Biogenesis of peroxisomes and mitochondria: linked by division. Histochem Cell Biol 131: Depreter M, Espeel M, Roels F Human peroxisomal disorders. Microsc Res Tech 61: Dodt G, Braverman N, Wong C, Moser A, Moser HW, Watkins P, Valle D, Gould SJ Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nat Genet 9: Dodt G, Gould SJ Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein import is mediated by a cycling receptor. J Cell Biol 135: Dodt G, Warren D, Becker E, Rehling P, Gould SJ. 2001a. Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p. J Biol Chem 276:

26 Chapter 1 1 Dodt G, Warren D, Becker E, Rehling P, Gould SJ. 2001b. Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p. J Biol Chem 276: Eaton J, Mouchou M Acatalasemia. In: Scriver C, Sly A, Beaudet W, Valle D, editors. The metabolic and molecular basis of inherited disease. New York: McGraw-Hill. p Ebberink MS, Mooyer PA, Koster J, Dekker CJ, Eyskens FJ, Dionisi-Vici C, Clayton PT, Barth PG, Wanders RJ, Waterham HR Genotype-phenotype correlation in PEX5-deficient peroxisome biogenesis defective cell lines. Hum Mutat 30: Eckert JH, Johnsson N Pex10p links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome. J Cell Sci 116: Einwachter H, Sowinski S, Kunau WH, Schliebs W Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway. EMBO Rep 2: Eitzen GA, Szilard RK, Rachubinski RA Enlarged peroxisomes are present in oleic acid-grown Yarrowia lipolytica overexpressing the PEX16 gene encoding an intraperoxisomal peripheral membrane peroxin. J Cell Biol 137: Elgersma Y, van den Berg M, Tabak HF, Distel B An efficient positive selection procedure for the isolation of peroxisomal import and peroxisome assembly mutants of Saccharomyces cerevisiae. Genetics 135: Elgersma Y, Vos A, van den Berg M, van Roermund CW, van der Sluijs P, Distel B, Tabak HF Analysis of the carboxyl-terminal peroxisomal targeting signal 1 in a homologous context in Saccharomyces cerevisiae. J Biol Chem 271: Erdmann R, Veenhuis M, Mertens D, Kunau WH Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 86: Erdmann R, Blobel G Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p. J Cell Biol 128: Faber KN, Heyman JA, Subramani S Two AAA family peroxins, PpPex1p and PpPex6p, interact with each other in an ATP-dependent manner and are associated with different subcellular membranous structures distinct from peroxisomes. Mol Cell Biol 18: Fagarasanu A, Fagarasanu M, Rachubinski RA Maintaining peroxisome populations: a story of division and inheritance. Annu Rev Cell Dev Biol 23: Fang Y, Morrell JC, Jones JM, Gould SJ PEX3 functions as a PEX19 docking factor in the import of class I peroxisomal membrane proteins. J Cell Biol 164: Ferdinandusse S, Denis S, Clayton PT, Graham A, Rees JE, Allen JT, McLean BN, Brown AY, Vreken P, Waterham HR, Wanders RJ Mutations in the gene encoding peroxisomal alpha-methylacyl- CoA racemase cause adult-onset sensory motor neuropathy. Nat Genet 24: Ferdinandusse S, Denis S, Mooijer PA, Zhang Z, Reddy JK, Spector AA, Wanders RJ Identification of the peroxisomal beta-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid. J Lipid Res 42: Ferdinandusse S, Ylianttila MS, Gloerich J, Koski MK, Oostheim W, Waterham HR, Hiltunen JK, Wanders RJ, Glumoff T. 2006a. Mutational spectrum of D-bifunctional protein deficiency and structure-based genotype-phenotype analysis. Am J Hum Genet 78: Ferdinandusse S, Kostopoulos P, Denis S, Rusch H, Overmars H, Dillmann U, Reith W, Haas D, Wanders RJ, Duran M, Marziniak M. 2006b. Mutations in the gene encoding peroxisomal sterol carrier protein X (SCPx) cause leukencephalopathy with dystonia and motor neuropathy. Am J Hum Genet 78: Ferdinandusse S, Denis S, Hogenhout EM, Koster J, van Roermund CW, Ijlst L, Moser AB, Wanders RJ, Waterham HR Clinical, biochemical, and mutational spectrum of peroxisomal acyl-coenzyme A oxidase deficiency. Hum Mutat 28: Fransen M, Wylin T, Brees C, Mannaerts GP, Van Veldhoven PP Human pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. Mol Cell Biol 21: Fransen M, Vastiau I, Brees C, Brys V, Mannaerts GP, Van Veldhoven PP Potential role for Pex19p in assembly of PTS-receptor docking complexes. J Biol Chem 279: Fransen M, Vastiau I, Brees C, Brys V, Mannaerts GP, Van Veldhoven PP Analysis of human Pex19p s domain structure by pentapeptide scanning mutagenesis. J Mol Biol 346: Fujiki Y, Matsuzono Y, Matsuzaki T, Fransen M Import of peroxisomal membrane proteins: the 24

27 Introduction interplay of Pex3p- and Pex19p-mediated interactions. Biochim Biophys Acta 1763: Gandre-Babbe S, van der Bliek AM The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells. Mol Biol Cell 19: Gatto GJJr, Geisbrecht BV, Gould SJ, Berg JM Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct Biol 7: Ghaedi K, Tamura S, Okumoto K, Matsuzono Y, Fujiki Y The peroxin pex3p initiates membrane assembly in peroxisome biogenesis. Mol Biol Cell 11: Glover JR, Andrews DW, Rachubinski RA Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer. Proc Natl Acad Sci U S A 91: Goldfischer S, Moore CL, Johnson AB, Spiro AJ, Valsamis MP, Wisniewski HK, Ritch RH, Norton WT, Rapin I, Gartner LM Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science 182: Gootjes J, Schmohl F, Mooijer PA, Dekker C, Mandel H, Topcu M, Huemer M, Von Schütz M, Marquardt T, Smeitink JA, Waterham HR, Wanders RJ Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism. Hum Mutat 24: Gould SJ, McCollum D, Spong AP, Heyman JA, Subramani S Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly. Yeast 8: Gould SJ, Kalish JE, Morrell JC, Bjorkman J, Urquhart AJ, Crane DI Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor. J Cell Biol 135: Gould SJ, Valle D Peroxisome biogenesis disorders: genetics and cell biology. Trends Genet 16: Gould SJ, Raymond GV, Valle D. 2001a. The peroxisome biogenesis disorders. In: Scriver C.R., Beaudet A.L., Sly W.S., Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill, Inc. p Gould SJ, Raymond GV, Valle D. 2001b. The peroxisome biogenesis disorders. In: Scriver C.R., Beaudet A.L., Sly W.S., Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill, Inc. p Gould SJ, Collins CS Opinion: peroxisomal-protein import: is it really that complex? Nat Rev Mol Cell Biol 3: Gouveia AM, Reguenga C, Oliveira ME, Sa-Miranda C, Azevedo JE Characterization of peroxisomal Pex5p from rat liver. Pex5p in the Pex5p-Pex14p membrane complex is a transmembrane protein. J Biol Chem 275: Gouveia AM, Guimaraes CP, Oliveira ME, Reguenga C, Sa-Miranda C, Azevedo JE Characterization of the peroxisomal cycling receptor Pex5p import pathway. Adv Exp Med Biol 544: Grou CP, Carvalho AF, Pinto MP, Wiese S, Piechura H, Meyer HE, Warscheid B, Sa-Miranda C, Azevedo JE Members of the E2D (UbcH5) Family Mediate the Ubiquitination of the Conserved Cysteine of Pex5p, the Peroxisomal Import Receptor. J Biol Chem 283: Grou CP, Carvalho AF, Pinto MP, Alencastre IS, Rodrigues TA, Freitas MO, Francisco T, Sa-Miranda C, Azevedo JE The peroxisomal protein import machinery--a case report of transient ubiquitination with a new flavor. Cell Mol Life Sci 66: Halbach A, Rucktaschel R, Rottensteiner H, Erdmann R The N-domain of Pex22p can functionally replace the Pex3p N-domain in targeting and peroxisome formation. J Biol Chem 284: Hettema EH, Motley AM How peroxisomes multiply. J Cell Sci 122: Heymans HS, Schutgens RB, Tan R, van den Bosch H, Borst P Severe plasmalogen deficiency in tissues of infants without peroxisomes (Zellweger syndrome). Nature 306: Honsho M, Tamura S, Shimozawa N, Suzuki Y, Kondo N, Fujiki Y. 1998a. Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D. Am J Hum Genet 63: Honsho M, Tamura S, Shimozawa N, Suzuki Y, Kondo N, Fujiki Y. 1998b. Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D. Am J Hum Genet 63: Honsho M, Fujiki Y Topogenesis of peroxisomal membrane protein requires a short, positively charged intervening-loop sequence and flanking hydrophobic segments. study using human membrane protein PMP34. J Biol Chem 276:

28 Chapter 1 1 Honsho M, Hiroshige T, Fujiki Y The membrane biogenesis peroxin Pex16p. Topogenesis and functional roles in peroxisomal membrane assembly. J Biol Chem 277: Huybrechts SJ, Van Veldhoven PP, Brees C, Mannaerts GP, Los GV, Fransen M Peroxisome dynamics in cultured mammalian cells. Traffic 10: Iwashita S, Tsuchida M, Tsukuda M, Yamashita Y, Emi Y, Kida Y, Komori M, Kashiwayama Y, Imanaka T, Sakaguchi M Multiple organelle-targeting signals in the N-terminal portion of peroxisomal membrane protein PMP70. J Biochem. Jansen GA, Wanders RJ, Watkins PA, Mihalik SJ Phytanoyl-coenzyme A hydroxylase deficiency -- the enzyme defect in Refsum s disease. N Engl J Med 337: Jansen GA, Hogenhout EM, Ferdinandusse S, Waterham HR, Ofman R, Jakobs C, Skjeldal OH, Wanders RJ Human phytanoyl-coa hydroxylase: resolution of the gene structure and the molecular basis of Refsum s disease. Hum Mol Genet 9: Jones JM, Morrell JC, Gould SJ Multiple distinct targeting signals in integral peroxisomal membrane proteins. J Cell Biol 153: Jones JM, Morrell JC, Gould SJ PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins. J Cell Biol 164: Kaur N, Hu J Dynamics of peroxisome abundance: a tale of division and proliferation. Curr Opin Plant Biol 12: Kelley RI, Datta NS, Dobyns WB, Hajra AK, Moser AB, Noetzel MJ, Zackai EH, Moser HW Neonatal adrenoleukodystrophy: new cases, biochemical studies, and differentiation from Zellweger and related peroxisomal polydystrophy syndromes. Am J Med Genet 23: Kemp S, Wanders RJ X-linked adrenoleukodystrophy: very long-chain fatty acid metabolism, ABC half-transporters and the complicated route to treatment. Mol Genet Metab 90: Kim PK, Mullen RT, Schumann U, Lippincott-Schwartz J The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER. J Cell Biol 173: Klein AT, van den Berg M, Bottger G, Tabak HF, Distel B Saccharomyces cerevisiae acyl-coa oxidase follows a novel, non-pts1, import pathway into peroxisomes that is dependent on Pex5p. J Biol Chem 277: Kobayashi S, Tanaka A, Fujiki Y Fis1, DLP1, and Pex11p coordinately regulate peroxisome morphogenesis. Exp Cell Res 313: Koch A, Schneider G, Luers GH, Schrader M Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1. J Cell Sci 117: Koch A, Yoon Y, Bonekamp NA, McNiven MA, Schrader M A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells. Mol Biol Cell 16: Koller A, Snyder WB, Faber KN, Wenzel TJ, Rangell L, Keller GA, Subramani S Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane. J Cell Biol 146: Lazarow PB, Fujiki Y Biogenesis of peroxisomes. Annu Rev Cell Biol 1: Lazarow PB Peroxisome biogenesis: advances and conundrums. Curr Opin Cell Biol 15: Li X, Baumgart E, Morrell JC, Jimenez-Sanchez G, Valle D, Gould SJ. 2002a. PEX11 beta deficiency is lethal and impairs neuronal migration but does not abrogate peroxisome function. Mol Cell Biol 22: Li X, Baumgart E, Dong GX, Morrell JC, Jimenez-Sanchez G, Valle D, Smith KD, Gould SJ. 2002b. PEX11alpha is required for peroxisome proliferation in response to 4-phenylbutyrate but is dispensable for peroxisome proliferator-activated receptor alpha-mediated peroxisome proliferation. Mol Cell Biol 22: Lingard MJ, Gidda SK, Bingham S, Rothstein SJ, Mullen RT, Trelease RN Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A cooperate in cell cycleassociated replication of peroxisomes. Plant Cell 20: Liu H, Tan X, Veenhuis M, McCollum D, Cregg JM An efficient screen for peroxisome-deficient mutants of Pichia pastoris. J Bacteriol 174: Marshall PA, Krimkevich YI, Lark RH, Dyer JM, Veenhuis M, Goodman JM Pmp27 promotes peroxisomal proliferation. J Cell Biol 129: Matsumura T, Otera H, Fujiki Y Disruption of the interaction of the longer isoform of Pex5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals. Study with a novel Pex5-impaired Chinese hamster ovary cell mutant. J Biol Chem 275: Matsuzaki T and Fujiki Y The peroxisomal membrane protein import receptor PEX3p is directly 26

29 Introduction transported to peroxisomes by a novel Pex19p- and Pex16-dependent pathway. J Cell Biol 183(7): Matsuzono Y, Kinoshita N, Tamura S, Shimozawa N, Hamasaki M, Ghaedi K, Wanders RJ, Suzuki Y, Kondo N, Fujiki Y Human PEX19: cdna cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly. Proc Natl Acad Sci U S A 96: Matsuzono Y, Matsuzaki T, Fujiki Y Functional domain mapping of peroxin Pex19p: interaction with Pex3p is essential for function and translocation. J Cell Sci 119: Moser HW, Smith KD, Watkins PA, Power J, Moser AB X-Linked Adrenoleukodystrophy. In: Scriver C.R., Beaudet A.L., Sly W.S., Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. New York: Mc Graw-Hill. p Motley AM, Brites P, Gerez L, Hogenhout E, Haasjes J, Benne R, Tabak HF, Wanders RJ, Waterham HR. 2002a. Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1. Am J Hum Genet 70: Motley AM, Brites P, Gerez L, Hogenhout E, Haasjes J, Benne R, Tabak HF, Wanders RJ, Waterham HR. 2002b. Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1. Am J Hum Genet 70: Munck JM, Motley AM, Nuttall JM, Hettema EH A dual function for Pex3p in peroxisome formation and inheritance. J Cell Biol 187: Muntau AC, Mayerhofer PU, Paton BC, Kammerer S, Roscher AA Defective peroxisome membrane synthesis due to mutations in human PEX3 causes Zellweger syndrome, complementation group G. Am J Hum Genet 67: Oliveira ME, Gouveia AM, Pinto RA, Sa-Miranda C, Azevedo JE The energetics of Pex5p-mediated peroxisomal protein import. J Biol Chem 278: Opperdoes FR, Borst P, Bakker S, Leene W Localization of glycerol-3-phosphate oxidase in the mitochondrion and particulate NAD+-linked glycerol-3-phosphate dehydrogenase in the microbodies of the bloodstream form to Trypanosoma brucei. Eur J Biochem 76: Otera H, Harano T, Honsho M, Ghaedi K, Mukai S, Tanaka A, Kawai A, Shimizu N, Fujiki Y The mammalian peroxin Pex5pL, the longer isoform of the mobile peroxisome targeting signal (PTS) type 1 transporter, translocates the Pex7p.PTS2 protein complex into peroxisomes via its initial docking site, Pex14p. J Biol Chem 275: Otera H, Setoguchi K, Hamasaki M, Kumashiro T, Shimizu N, Fujiki Y Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import. Mol Cell Biol 22: Pinto MP, Grou CP, Alencastre IS, Oliveira ME, Sa-Miranda C, Fransen M, Azevedo JE The import competence of a peroxisomal membrane protein is determined by Pex19p before the docking step. J Biol Chem 281: Pinto MP, Grou CP, Fransen M, Sa-Miranda C, Azevedo JE The cytosolic domain of PEX3, a protein involved in the biogenesis of peroxisomes, binds membrane lipids. Biochim Biophys Acta 1793: Platta HW, El MF, Schlee D, Grunau S, Girzalsky W, Erdmann R Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling. J Cell Biol 177: Platta HW, El MF, Baumer BE, Schlee D, Girzalsky W, Erdmann R Pex2 and pex12 function as protein-ubiquitin ligases in peroxisomal protein import. Mol Cell Biol 29: Poll-The BT, Saudubray JM, Ogier HA, Odievre M, Scotto JM, Monnens L, Govaerts LC, Roels F, Cornelis A, Schutgens RB, Infantile Refsum disease: an inherited peroxisomal disorder. Comparison with Zellweger syndrome and neonatal adrenoleukodystrophy. Eur J Pediatr 146: Poll-The BT, Roels F, Ogier H, Scotto J, Vamecq J, Schutgens RB, Wanders RJ, van Roermund CW, van Wijland MJ, Schram AW, A new peroxisomal disorder with enlarged peroxisomes and a specific deficiency of acyl-coa oxidase (pseudo-neonatal adrenoleukodystrophy). Am J Hum Genet 42: Purdue PE, Yang X, Lazarow PB Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway. J Cell Biol 143: Rhodin J Correlation of ultrastructural organization in normal and experimentally changed proximal convoluted tubule cells of the mouse kidney. PhD Thesis, Karolinska Institutet Stockholm, Sweden. Rottensteiner H, Kramer A, Lorenzen S, Stein K, Landgraf C, Volkmer-Engert R, Erdmann R

30 Chapter 1 1 Peroxisomal membrane proteins contain common Pex19p-binding sites that are an integral part of their targeting signals. Mol Biol Cell 15: Sacksteder KA, Gould SJ The genetics of peroxisome biogenesis. Annu Rev Genet 34: Sacksteder KA, Jones JM, South ST, Li X, Liu Y, Gould SJ PEX19 binds multiple peroxisomal membrane proteins, is predominantly cytoplasmic, and is required for peroxisome membrane synthesis. J Cell Biol 148: Saidowsky J, Dodt G, Kirchberg K, Wegner A, Nastainczyk W, Kunau WH, Schliebs W The diaromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14. J Biol Chem 276: Schneiter R, Brugger B, Sandhoff R, Zellnig G, Leber A, Lampl M, Athenstaedt K, Hrastnik C, Eder S, Daum G, Paltauf F, Wieland FT, Kohlwein SD Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane. J Cell Biol 146: Schrader M, Reuber BE, Morrell JC, Jimenez-Sanchez G, Obie C, Stroh TA, Valle D, Schroer TA, Gould SJ Expression of PEX11beta mediates peroxisome proliferation in the absence of extracellular stimuli. J Biol Chem 273: Serasinghe MN, Yoon Y The mitochondrial outer membrane protein hfis1 regulates mitochondrial morphology and fission through self-interaction. Exp Cell Res 314: Shimozawa N, Suzuki Y, Zhang Z, Imamura A, Ghaedi K, Fujiki Y, Kondo N Identification of PEX3 as the gene mutated in a Zellweger syndrome patient lacking peroxisomal remnant structures. Hum Mol Genet 9: Shimozawa N, Nagase T, Takemoto Y, Suzuki Y, Fujiki Y, Wanders RJ, Kondo N A novel aberrant splicing mutation of the PEX16 gene in two patients with Zellweger syndrome. Biochem Biophys Res Commun 292: Sichting M, Schell-Steven A, Prokisch H, Erdmann R, Rottensteiner H Pex7p and Pex20p of Neurospora crassa function together in PTS2-dependent protein import into peroxisomes. Mol Biol Cell 14: Sindelar PJ, Guan Z, Dallner G, Ernster L The protective role of plasmalogens in iron-induced lipid peroxidation. Free Radic Biol Med 26: Snyder WB, Koller A, Choy AJ, Subramani S The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane. J Cell Biol 149: Soukupova M, Sprenger C, Gorgas K, Kunau WH, Dodt G Identification and characterization of the human peroxin PEX3. Eur J Cell Biol 78: South ST, Gould SJ Peroxisome synthesis in the absence of preexisting peroxisomes. J Cell Biol 144: Stanley WA, Filipp FV, Kursula P, Schuller N, Erdmann R, Schliebs W, Sattler M, Wilmanns M Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p. Mol Cell 24: Stanley WA, Fodor K, Marti-Renom MA, Schliebs W, Wilmanns M Protein translocation into peroxisomes by ring-shaped import receptors. FEBS Lett 581: Steinberg SJ, Dodt G, Raymond GV, Braverman NE, Moser AB, Moser HW Peroxisome biogenesis disorders. Biochim Biophys Acta 1763: Swinkels BW, Gould SJ, Bodnar AG, Rachubinski RA, Subramani S A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J 10: Tabak HF, Murk JL, Braakman I, Geuze HJ Peroxisomes start their life in the endoplasmic reticulum. Traffic 4: Tabak HF, Hoepfner D, Zand A, Geuze HJ, Braakman I, Huynen MA Formation of peroxisomes: present and past. Biochim Biophys Acta 1763: Titorenko VI, Chan H, Rachubinski RA Fusion of small peroxisomal vesicles in vitro reconstructs an early step in the in vivo multistep peroxisome assembly pathway of Yarrowia lipolytica. J Cell Biol 148: Urquhart AJ, Kennedy D, Gould SJ, Crane DI Interaction of Pex5p, the type 1 peroxisome targeting signal receptor, with the peroxisomal membrane proteins Pex14p and Pex13p. J Biol Chem 275: Van Ael E, Fransen M Targeting signals in peroxisomal membrane proteins. Biochim Biophys Acta 1763:

31 Introduction van den Bosch H, Schutgens RB, Wanders RJ, Tager JM Biochemistry of peroxisomes. Annu Rev Biochem 61: Van der Leij I, van den Berg M, Boot R, Franse M, Distel B, Tabak HF Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure. J Cell Biol 119: Walton PA, Hill PE, Subramani S Import of stably folded proteins into peroxisomes. Mol Biol Cell 6: Wanders RJ, Schumacher H, Heikoop J, Schutgens RB, Tager JM Human dihydroxyacetonephosphate acyltransferase deficiency: a new peroxisomal disorder. J Inherit Metab Dis 15: Wanders RJ, Dekker C, Hovarth VA, Schutgens RB, Tager JM, Van Laer P, Lecoutere D Human alkyldihydroxyacetonephosphate synthase deficiency: a new peroxisomal disorder. J Inherit Metab Dis 17: Wanders RJ, Vreken P, Ferdinandusse S, Jansen GA, Waterham HR, van Roermund CW, Van Grunsven EG. 2001a. Peroxisomal fatty acid alpha- and beta-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem Soc Trans 29: Wanders RJ, Jansen GA, Lloyd MD Phytanic acid alpha-oxidation, new insights into an old problem: a review. Biochim Biophys Acta 1631: Wanders RJ, Waterham HR Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders. Clin Genet 67: Wanders RJ, Waterham HR. 2006a. Biochemistry of mammalian peroxisomes revisited. Annu Rev Biochem 75: Wanders RJ, Waterham HR. 2006b. Peroxisomal disorders: the single peroxisomal enzyme deficiencies. Biochim Biophys Acta 1763: Wanders R, Jakobs C, Skjeldal O. 2001b. Refsum disease. In: Scriver C, Beaudet A, Sly W, Valle D, editors. The metabolic & molecular bases of inherited disease. New York: Mc Graw-Hill. p Wang X, Unruh MJ, Goodman JM Discrete targeting signals direct Pmp47 to oleate-induced peroxisomes in Saccharomyces cerevisiae. J Biol Chem 276: Wang X, McMahon MA, Shelton SN, Nampaisansuk M, Ballard JL, Goodman JM Multiple targeting modules on peroxisomal proteins are not redundant: discrete functions of targeting signals within Pmp47 and Pex8p. Mol Biol Cell 15: Waterham HR, Titorenko VI, Swaving GJ, Harder W, Veenhuis M Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles. EMBO J 12: Waterham HR, Koster J, van Roermund CW, Mooyer PA, Wanders RJ, Leonard JV A lethal defect of mitochondrial and peroxisomal fission. N Engl J Med 356: Watkins PA, Chen WW, Harris CJ, Hoefler G, Hoefler S, Blake DC, Jr., Balfe A, Kelley RI, Moser AB, Beard ME, Peroxisomal bifunctional enzyme deficiency. J Clin Invest 83: Weller S, Gould SJ, Valle D Peroxisome biogenesis disorders. Annu Rev Genomics Hum Genet 4: Williams C, van den Berg M, Geers E, Distel B Pex10p functions as an E3 ligase for the Ubc4pdependent ubiquitination of Pex5p. Biochem Biophys Res Commun 374: Williams EL, Acquaviva C, Amoroso A, Chevalier F, Coulter-Mackie M, Monico CG, Giachino D, Owen T, Robbiano A, Salido E, Waterham H, Rumsby G Primary hyperoxaluria type 1: update and additional mutation analysis of the AGXT gene. Hum Mutat 30: Yahraus T, Braverman N, Dodt G, Kalish JE, Morrell JC, Moser HW, Valle D, Gould SJ The peroxisome biogenesis disorder group 4 gene, PXAAA1, encodes a cytoplasmic ATPase required for stability of the PTS1 receptor. EMBO J 15: Yang X, Purdue PE, Lazarow PB Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p. Eur J Cell Biol 80: Zoeller RA, Lake AC, Nagan N, Gaposchkin DP, Legner MA, Lieberthal W Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether. Biochem J 338 ( Pt 3):

32 Chapter

33 Chapter 2 Genetic Classification and Mutational Spectrum of More Than 600 Patients with a Zellweger Syndrome Spectrum Disorder Merel S. Ebberink, Petra A.W. Mooijer, Jeannette Gootjes, Janet Koster, Ronald J.A. Wanders, Hans R. Waterham Laboratory Genetic Metabolic Diseases, Academic Medical Centre at the University of Amsterdam, Amsterdam, the Netherlands. Submitted for publication

34 Chapter 2 Abstract 2 Introduction: The autosomal recessive Zellweger syndrome spectrum (ZSS) disorders comprise a main subgroup of the peroxisome biogenesis disorders. The ZSS disorders can be caused by mutations in any of 12 different currently identified PEX genes resulting in severe, often lethal, multi-systemic disorders. Objective: To get insight into the spectrum of PEX gene defects among ZSS disorders and to investigate if additional human PEX genes are required for functional peroxisome biogenesis, we assigned over 600 ZSS fibroblast cell lines to different genetic complementation groups. Methods: Fibroblast cell lines were subjected to a complementation assay involving fusion by means of polyethylene glycol or a PEX cdna transfection assay specifically developed for this purpose. For the majority of the cell lines we subsequently determined the underlying mutations by sequence analysis of the implicated PEX genes. Results and Discussion: The PEX cdna transfection assay allows for the rapid identification of PEX genes defective in ZSS patients. The assignment of over 600 fibroblast cell lines to different genetic complementation groups provides the most comprehensive and representative overview of the frequency distribution of the different PEX gene defects. We did not identify any novel genetic complementation group, suggesting that all PEX gene defects resulting in peroxisome deficiency are currently known. 32

35 Genetic Classification and Mutational Spectrum of ZSS Introduction Peroxisome biogenesis disorders (MIM ) include the Zellweger syndrome spectrum (ZSS) disorders and Rhizomelic Chondrodysplasia Punctata type I (Weller et al., 2003). The latter is caused by mutations in the PEX7 gene, encoding a cytosolic receptor involved in the import of a small subset of peroxisomal matrix proteins (Braverman et al., 2002; Motley et al., 2002). The ZSS disorders include the Zellweger syndrome (ZS, MIM ), neonatal adrenoleukodystrophy (NALD, MIM ) and infantile Refsum disease (IRD, MIM ), which represent a spectrum of disease severity with ZS being the most, and IRD the least severe disorder. ZSS disorders are autosomal recessive disorders and can be caused by a defect in any of at least 12 different PEX genes (Steinberg et al., 2006). These PEX genes encode proteins called peroxins that are involved in various stages of peroxisomal protein import and/or the biogenesis of peroxisomes. Severe defects in one of these PEX genes result in a complete absence of functional peroxisomes as observed in the Zellweger Syndrome phenotype. The NALD or IRD phenotypes are often associated with residual peroxisomal functions and/or partial functional peroxisomes (i.e. peroxisomal mosaicism; Wanders and Waterham, 2006; Weller et al., 2003)). Many human PEX genes have been identified on the basis of sequence similarity with yeast PEX genes identified by functional complementation of peroxisomedeficient yeast mutants. At present more than 30 different yeast peroxins have been identified that are involved in different peroxisomal processes, including the formation of peroxisomal membranes, peroxisomal growth, fission and proliferation, and import of matrix proteins (Platta and Erdmann, 2007). Of these yeast peroxins, 19 have been implicated in peroxisome protein import and biogenesis, whereas in humans, so far only 13 peroxins have been identified that are required for these processes. To study whether human peroxisome biogenesis requires the action of additional peroxins, we set out to assign primary skin fibroblasts from patients diagnosed with a ZSS disorder to different genetic complementation groups. Following the complementation analysis, we performed mutation analysis of the respective PEX genes in most of the cell lines. We here report an overview of our genetic complementation studies in more than 600 patient cell lines with a Zellweger syndrome spectrum disorder. In addition, we provide an overview of all mutations we identified in these patient cell lines, completed with previously reported mutations in the respective PEX genes. 2 Methods Cell lines For this study, we used 613 independent primary skin fibroblast cell lines from non-related patients that had been sent to our laboratory since 1985 for diagnostic evaluation and which were diagnosed with a ZSS disorder. The diagnosis is based on metabolite analysis in plasma, which ideally involves analysis of very long chain fatty acids (VLCFAs), bile acid intermediates, phytanic acid, pristanic acid, pipecolic acid and plasmalogens, and/or detailed studies in fibroblasts, including 33

36 Chapter 2 2 VLCFA analysis, C26:0 and pristanic acid β-oxidation, phytanic acid α-oxidation and dihydroxyacetonephosphate acyltransferase activity analysis, and the absence of peroxisomes assessed by catalase immunofluorescence (IF) microscopy (Wanders and Waterham, 2005). The cell lines were cultured in DMEM medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Bio-Whittaker), 100 U/ ml penicillin, 100 µg/ml streptomycin and 25 mm Hepes buffer with L-glutamine in a humidified atmosphere of 5% CO 2 and at 37 C. Genetic complementation assays To assign cell lines to genetic complementation groups, we used a previously described polyethylene glycol (PEG)- mediated cell fusion assay (Brul et al., 1988) and a PEX cdna transfection assay, specifically developed for this purpose. In the latter assay, ZSS cell lines are co-transfected separately with the different PEX cdnas subcloned in the pcdna3 expression vector and an egfp-skl vector (Waterham et al., 2007). The egfp-skl vector expresses egfp tagged with the peroxisomal targeting signal -SKL, which allows immediate visual detection of peroxisomes when present. The transfections were performed with the NHDF Nucleofector Kit (Amaxa, Cologne, Germany) and the Nucleofector Device (Amaxa, Cologne, Germany) using the standard program U-23, which in our hands results in high transfection efficiencies ranging from 70-80% for primary skin fibroblasts. We used the following transfection order of PEX expression vectors based on the estimated frequency of the different PEX gene defects: 1) PEX1, PEX6 and PEX12; 2) PEX2, PEX10 and PEX26; 3) PEX5, PEX13 and PEX14. Transfection with PEX3, PEX16 and PEX19 was performed when the patient fibroblasts show no peroxisomal membrane remnants based on immunofluorescence microscopy with antibodies against ALDP, a peroxisomal membrane protein. Cells were examined by means of fluorescence microscopy 48 to 72 hours after transfection to determine the subcellular localization of the peroxisomal reporter protein egfp-skl. Mutation analysis Mutation analysis was performed by either sequencing all exons plus flanking intronic sequences of the PEX genes amplified by PCR from genomic DNA or by sequencing PEX cdnas prepared from total RNA fractions. Genomic DNA was isolated from skin fibroblasts using the NucleoSpin Tissue genomic DNA purification kit (Macherey-nagel, Germany, Düren). Total RNA was isolated from skin fibroblasts using Trizol (Invitrogen, Carlsbad, CA) extraction, after which cdna was prepared using a first strand cdna synthesis kit for RT-PCR (Roche, Mannheim, Germany). All forward and reverse primers (sequences available on request) were tagged with a -21M13 (5- TGTAAAACGACGGCCAGT-3 ) sequence or M13rev (5 -CAGGAAACAGCTATGACC-3 ) sequence, respectively. PCR fragments were sequenced in two directions using -21M13 and M13rev primers by means of BigDye Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA) and analyzed on an Applied Biosystems 377A automated DNA sequencer, following the manufacturer s protocol (Applied Biosystems, Foster City, CA, USA). Mutation analysis of PEX1 (GenBank accession number NM_000466) was performed by sequencing the coding region of the PEX1 cdna and/ or exons amplified from gdna and by RFLP analysis to identify the frequently occurring p.g843d mutation. Mutations 34

37 Genetic Classification and Mutational Spectrum of ZSS in the PEX2 gene were identified by sequencing exon 4 from gdna (NM_000318). The PEX3 (NM_003630) gene was analyzed by sequencing of the coding region of the PEX3 cdna (NM_003630). Mutation analysis of PEX5L (X84899), PEX10 (NM_002617), PEX12 (NM_000286), PEX13 (NM_002618), PEX14 (NM_004565), PEX16 (NM_004813), PEX19 (NM_002857) and PEX26 (NM_017929) were performed by sequencing of all exons plus flanking intronic sequences amplified from gdna. Sequences were compared to the indicated reference sequences with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. 2 Results and Discussion Genetic complementation Initially, we used a previously reported cell fusion complementation assay (Brul et al., 1988). Because this assay is rather laborious, we developed a more rapid and Figure 1. Complementation testing by PEG fusion and by PEX cdna transfection. Principle of the PEG fusion method. (A), a tester cell line with a known PEX gene defect and a patient cell line are fused by PEG treatment resulting in multi-nucleated cells. Cells are examined by catalase immunofluorescence microscopy to determine complementation (B) or non complementation (C). Principle of the PEX cdna transfection assay. The pcdna3-pex expression vectors are co-transfected separately but together with egfp-skl into patient cells (D). Cells are examined by direct fluorescent microscopy to determine complementation (E) or non complementation (F). 35

38 Chapter 2 2 direct complementation assay taking advantage of the known identities of the 12 human PEX genes that can cause a ZSS disorder (for principle see Figure 1). The PEX cdna transfection assay worked very well for cell lines displaying complete peroxisome deficiencies, and for cell lines displaying different degrees of peroxisomal mosaicism that are sensitive to culturing at 40ºC, which exacerbates the defect in peroxisome biogenesis (Gootjes et al., 2004d). We assigned 613 different ZSS cell lines to genetic complementation groups (Table 1). The results give a representative overview of the spectrum of PEX gene defects among ZSS disorders with PEX1 (MIM602136), PEX6 (MIM601498) and PEX12 (MIM601758) defects being the most common. Although we received ZSS cell lines from different parts of the world, they predominantly (>90%) are of Caucasian origin and thus the spectrum of PEX gene defects primarily relates to the Caucasian race. Table 1. Frequency distribution of PEX gene defects in ZSS. Classification of the 613 different patient cell lines was done by either genetic complementation (cdna transfection of PEG fusion) or by sequence analysis. PEX gene Genetic complementation (number of cell lines) Frequency (%) PEX PEX PEX3 3 <1 PEX PEX PEX PEX PEX PEX14 3 <1 PEX PEX19 4 <1 PEX Somewhat unexpected, all the analyzed ZSS cell lines could be assigned to one of the known genetic complementation groups and no novel complementation group was identified. In a recent study, where 90 cell lines were assigned to genetic complementation groups using a similar approach, also no novel complementation groups were identified (Krause et al., 2009). This suggests that, in contrast to yeast where 19 different PEX genes have been implicated, peroxisome protein import and biogenesis in humans would only require 12 PEX genes. However, it should be emphasized that to be suitable for genetic complementation testing, cell lines need to display a complete peroxisome deficiency in the majority of cells. It therefore remains possible that defects in other putative human PEX genes cannot be identified by this approach, because they don t result in peroxisome deficiency or perhaps lead to different clinical presentations, as recently exemplified by a very distinct peroxisomal and mitochondrial fission defect due to a dominant negative mutation in the DLP1 36

39 Genetic Classification and Mutational Spectrum of ZSS gene (Waterham et al., 2007). In addition, defects in certain PEX genes may also be without consequences due to gene redundancy. For example, the yeast PEX4 gene, which is associated with complete peroxisome deficiency in yeast, appears to have 3 human ortholoques (Grou et al., 2008). Considering that these ortholoques are encoded by different genes and each can promote the ubiquitination of PEX5 (MIM600414) on its own, it seems unlikely that a defect in one of these proteins will cause a peroxisomal disorder (Grou et al., 2008). Mutation analysis After the assignment of patient cell lines to genetic complementation groups, we performed mutation analysis of the respective PEX genes in the majority of the cell lines. We here report the mutations we identified in the different PEX genes. The reported mutations are assumed to be pathogenic for the following reasons: 1) They are the only sequence variants identified in the coding regions or flanking intronic sequences of the different PEX genes, which were demonstrated to be defective by the fact that peroxisome biogenesis in the cell line is restored after transfection with the respective PEX cdna (i.e. functional assay). 2) The sequence variants were not listed in the SNP database or encountered in more than 100 control chromosomes. It should be noted that in the vast majority of cell lines we identified either two heterozygous or one homozygous putative pathogenic mutation. In few cell lines we only found one heterozygous mutation after sequencing gdna, which in most cases appeared homozygously expressed when analyzing the respective PEX cdna, indicating that the second, unidentified mutation affects gene transcription or mrna stability. 2 PEX1 is the most affected gene among patients with a ZSS disorder. It encodes a protein belonging to the AAA ATPase protein family (ATPases Associated with various cellular Activities) and contains two ATP-binding folds. Table 2 lists all mutations identified in the cell lines we analyzed, completed with mutations reported in literature. Among these mutations a few common mutations have been identified (Maxwell et al., 2005). The most common mutation in PEX1 is a missense mutation in the second ATP-binding domain (c.2528g>a) leading to p.g843d. This mutation reduces the binding between PEX1 and PEX6 and is known for its temperature sensitivity, meaning that when fibroblasts of patients homozygous for G843D are cultured at 30 C, they regain the ability to import catalase and other peroxisomal matrix proteins, as well as the various peroxisomal metabolic functions (Imamura et al., 1998a; Maxwell et al., 2005). Patients homozygous for the G843D mutation usually present with the milder IRD phenotype. The second most common mutation in PEX1 is c.2079_2098inst, which results in a frame shift predicted to cause a truncated PEX1 protein (Collins and Gould, 1999), although Northern blot analysis showed that this mutation results in low steady state PEX1 mrna levels (Maxwell et al., 2005). Patients homozygous for this mutation are affected with the severe ZS phenotype. The c. 2916delA mutation is the third most common mutation in PEX1; this mutation leads to a frame shift. When combined with previously reported PEX1 mutations, a total of 81 different mutations has now been identified in the PEX1 gene, of which 32 have not been reported previously. 37

40 Chapter 2 Table 2. Mutations in the PEX1 gene. 2 Number of patients Mutations identified in this study Homozygouzygous Hetero- Exon Nucleotide Amino acid Reference 1 c.2t>c disruption startcodon 3 this study 1 c.3g>a disruption startcodon 1 this study 1 c.5g>a p.w2x 1 this study 1 c.56_57deltg p.v19gfsx48 1 this study 2 c.130-2a>t - 1 this study i2 c.273+1g>a - 1 this study 3 c.274g>c p.v92l (a) 3 c.343_344insct p.d115afsx18 1 this study 4 c.434_448delinsgcaa p.v145_ 1 (b) Q150delinsGfsX24 5 c.547c>t p.r183x 1 (c) 5 c.569c>a p.s190x 4 this study 5 c.616c>t p.q206x 1 this study 5 c.781c>t p.q261x (d) 5 c delaa p.q261rfsx8 3 (c) 5 c.788_789delca p.t263fsx5 1 (b) 5 c.904delg p.a302qfsx23 (a,e) 5 c.911_912delct p.s304fsx3 1 (m) 5 c.1007t>c p.v336a 1 this study 5 c.1108_1109insa p.i370nfsx2 1 (a) 5 c.1193delt p.i398tfsx7 1 this study i5 c g>t - 1 (c) 6 c.1340t>c p.l447s 1 this study 7 c.1411c>t p.q471x 1 this study 8 c.1501_1502delct p.l501x 1 this study 8 c.1509_1510dupta p.v504x 1 this study 8 c.1579a>g p.t526a 1 this study 9 c.1663t>c p.s555p 1 this study i9 c g>t - 1 (b) 10 c.1706a>c p.h569p 1 this study 10 c.1713_1716deltcac p.h571fsx10 1 this study 10 c.1716_1717delca p.h572qfsx19 2 (c) 10 c.1777g>a p.g593r 1 (b) 11 c.1842dela p.e615kfsx30 1 this study 11 c.1865_1866inscagtgtgga - (f) 11 c.1886_1887delgt p.c629x 1 this study 11 c.1891delg p.a631lfsx14 1 this study 11 c.1897c>t p.r633x (d) 12 r.1900_2070del p.g634_h690del (g) 12 c.1957t>a p.w653r 1 this study 12 c.1960_1961dupcagtgtgga p.t651_w653dup 1 (f,h,i,j) 12 c.1976t>a p.v659d 1 this study 12 c.1991t>c p.l664p (d) 12 c.2008c>a p.l670m 1 (b) 12 c.2034_2035delca p.h678qfsx15 1 (k) 12 c g>t - 5 (b) 38

41 Genetic Classification and Mutational Spectrum of ZSS Table 2. (continue) Mutation Number of patient identified in this study Exon Nucleotide Amino Acids Homozygouzygous Hetro- Reference 13 c.2083_2085delatg p.m659del (m) 13 c delgataa p.m695ifsx45 (k) 13 c.2097_2098inst p.i700fsx (a,b,f,k,l) 13 c.2224c>t p.q742x 2 this study 13 c.2227_2416del p.e743nfsx2fs (f) 14 c.2364g>a Deletion exon 14 1 this study 14 c.2368c>t p.r790x 1 (e) 14 c.2383c>t p.r795x 1 (l) 14 c.2387t>c p.l796p 1 (m) 14 c.2391_2392deltc p.r798sfsx35 (a) 14 c.2392c>g p.r798g 1 (e) 15 c.2528g>a p.g843d (b,e,f,k,l) 15 c.2537_2545delatgaagtta p.h846_ (k) instcatggt R849delinsLfsX bpinsex15 - (j) 16 c.2614c>t p.r872x 2 (f) 16 c.2633_2635 deltgt p.l879del 1 1 this study 16 c.2636t>c p.l879s 3 this study 16 c.2654c>g p.t885r 1 thid study 17 c.2730dela p.l910fsx (b) 17 c.2760dela p.a921lfsx40 1 (this study) 18 c.2814_2818delctttg p.f938lfsx2 (f) 18 c.2845c>t p.r949w 1 this study 18 c.2846g>a p.r949q 2 (b) 18 c.2916dela p.g973afsx (e,f,k) 18 c g>a - 1 (k) 18 c _2926+3inst - (l) 18 c t>c - (b) 19 c t_3208- Delition exon 19 and 2 this study 342AdelinsATAGTATAGA & c _ exon c.2966t>c p.i989t (b) 19 c.2992c>t p.r998x (e) 19 c.2993g>a p.r998q (a) 19 c.3022_3024delcct p.p1008del (k) 20 c.3038g>a p.r1013h (m) 20 c.3180_3181inst p.g1061wfsx16 (l) 20 c g>c - (l) 21 c.3287c>g p.s1096x (m) 21 - p.y1126x (f) 23 c delagtc p.q1231hfsx3 1 (c, m) 24 c.3850t>c p.x1284qnextx29 1 (b) (a) Maxwell et al., 2005 (f) Preuss et al., 2002 (k) Steinberg et al., 2004 (b) Walter et al., 2001 (g) Tamura et al., 1998 (l) Collins and Gould, 1999 (c) Yik et al., 2009 (h) Gartner et al., 1999 (m) Rosewich et al, 2005 (d) Tamura et al., 2001 (i) Portsteffen et al., 1997 (e) Maxwell et al., 2002 (j) Reuber et al.,

42 Chapter 2 2 The PEX2 (MIM ) gene was the first gene reported to be mutated in ZSS patients. PEX2 is an integral membrane protein with two transmembrane domains and a zinc-binding motif, probably involved in the poly-ubiquitination of the peroxisomal import receptor PEX5 (Platta et al., 2009). The PEX2 gene contains four exons, but the entire coding sequence is included in exon 4. One mutation in the PEX2 gene (c.355c>t) was found to be associated with temperature sensitivity: cells cultured at 30 C show partial catalase import (Imamura et al., 1998b). The patient fibroblasts homozygous for the c.669g>a mutation displayed a mosaic peroxisomal pattern, characterized by import competency of catalase in 30% of the cells (Gootjes et al., 2003; Gootjes et al., 2004c). In this study, six novel mutations have been identified, making a total of 18 different mutations currently identified in the PEX2 gene (Table 3). Table 3. Mutations in the PEX2 gene. Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygous Heterozygous Reference 4 c.115c>t p.q39x (b) 4 c.163 G>A p.e55k (a, c) 4 c.273delt p.n92tfsx2 (a) 4 c.279_283delgagat p.r94sfsx5 2 1 (d) 4 c.286c>t p.q96x 1 this study 4 c.325inst p.c109lfsx14 1 this study 4 c.339_345delcaggtgg p.r114x 1 1 this study 4 c.355c>t p.r119x 4 1 (a, c, e) 4 c.373c>t p.r125x (a, c, f) 4 c.550delc p.r184vfsx8 3 (g) 4 c.642delg p.k215sfsx2 1 (g) 4 c.669g>a p.w223x 2 (d) 4 c.739t>c p.c247r 1 (d) 4 c.782a>g p.h261r 1 this study 4 c.791g>a p.c264y 1 this study 4 c.834_838deltactt p.f278lfsx3 1 (b) 4 c.866insa p.s289fsx36 1 this study (a) Steinberg et al., 2004 (d) Gootjes et al., 2004a (g) Shimozawa et al., 2000b (b) Krause et al., 2006 (e) Shimozawa et al., 1993 (c) Imamura et al., 1998b (f) Shimozawa et al., 1998 PEX3 (MIM603164) is an integral membrane protein with two putative membranespanning domains. The exact function of PEX3 is unclear, but it is assumed that PEX3 plays a role in insertion of peroxisomal membrane proteins into the peroxisomal membrane based on the absence of peroxisomal ghosts in cell lines with a defect in PEX3 (Ghaedi et al., 2000; Muntau et al., 2000). In total, six different mutations in PEX3 were identified of which four have been reported previously (Table 4). 40

43 Genetic Classification and Mutational Spectrum of ZSS Table 4. Mutations in the PEX3 gene. Mutation Number of patients identified in this study Exon Nucleotide Amino acid Homozygous Heterozygous Reference 2 c.157c>t p.r53x (a) 4 c.328_331delataa p.i110vfsx23 1 this study 5 r.334_393del p.f112_v131del 1 this study 7 c.543_544inst p.v182cfsx2 (a, b, c) 10 c.856c>t p.r286x 1 (d) 11 c.942-8t>g p.s314rfsx3 (b, e) 2 (a) South et al., 2000 (c) Shimozawa et al., 2000a (e) Ghaedi et al., 2000 (b) Muntau et al., 2000 (d) Dursun et al., 2009 PEX5 is a protein with seven di-aromatic pentapeptide repeats (WxxxF/Y) essential for docking to the peroxisomal membrane and for binding to either PEX13 (MIM601789) or PEX14 (MIM601791, (Saidowsky et al., 2001; Weller et al., 2003) and with seven tetrapeptide repeats (TPRs) to mediate the interaction with the PTS1 sequence. In humans, two functional protein variants of PEX5 are produced as a result of alternative splicing of the PEX5 mrna. The longest variant, PEX5L, contains an additional 111bp encoding 37 amino acids, due to alternative splicing of exon 7 (Dodt et al., 2001). The shorter protein, PEX5S, has been reported to be exclusively involved in peroxisomal PTS1 protein import, whereas PEX5L mediates both PTS1 and PTS2 protein import. Recently, we published 11 mutations in the PEX5 gene (Ebberink et al., 2009). These mutations combined with previously reported mutations are listed in table 5. Table 5. Mutations in the PEX5 gene. Mutations Exon Nucleotide Amino acid Number of patients identified in this study Homozygous Heterozygous (a) Ebberink et al., 2009 (b) Dodt et al., 1995 (c ) Shimozawa et al., 1999b Reference 5 c.548_552-55delins238inv p.d150gfsx33 1 (a) 7 c.826c>t p.r276x 1 (a) 10 c.1090c>t p.q364x 1 (a) 11 [c.1244a>g]+[c.548- p.n415s 1 (a) 549dupATCG; c.604g>c] i12 c g>a p.r394s, r.395_465del 1 (a) 12 c.1258c>t p.r420x 2 (a) 12 c.1279c>t p.r427x (b) i14 c a>c r.1561_1718del, 1 (a) r.1561_1584del 14 c.1578t>g pn526k 2 (a, b) 14 c.1669c>t p.r557w 1 (a) 15 c.1799c>g p.s600w 1 (a, c) 41

44 Chapter 2 The PEX6 gene consists of 17 exons and mutations in the PEX6 gene were found to be scattered throughout the whole gene. PEX6 is also a member of the AAA ATPase family and interacts with PEX1. PEX6 is required for import of peroxisomal matrix proteins and/or vesicle fusion. At present, 77 different mutations in PEX6 gene have been identified, a complete overview of which has been reported very recently and thus will not been shown here (Ebberink et al., 2010a). 2 PEX10 (MIM602859) is an integral membrane protein and contains two transmembrane domains and a C-terminal zinc-binding motif, like PEX2 and PEX12. Two mrna splice forms of PEX10 have been identified, resulting from the use of a different splice acceptor site at the 3 end of intron 3. The longer form accounts for 10% of the PEX10 mrna in the cell and appears to be slightly less functional (Warren et al., 2000). Combined with previously reported PEX10 mutations, 26 different mutations have been identified in the PEX10 gene of which nine have not been reported previously (Table 6). Table 6. Mutations in the PEX10 gene. Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygous Heterozygous Reference 1 c.2t>c disruption startcodon 1 (a) 1 c.3g>a disruption startcodon 1 this study 1 c.4delg p.a2pfsx9 (b) 1 c.13_28del, ins20bp p.a5pfsx46 (c) 3 c.203c>a p.t68n 1 this study 3 c.208g>c p.g70r 1 this study 3 c.211g>a p.e71k 1 this study 3 c.233a>g p.q78r 1 this study 3 c.337delc p.l113wfsx40 (d) 3 c.352c>t p.q118x 1 this study 3 c.373c>t p.r125x (e) i3 c.600+1g>a - 1 (e) 4 c.697delg p.a213pfsx131 1 this study 4 c.704_705insa p.l236afsx (b, c) 4 c.646_647insa p.s216kfsx123 1 this study 4 c.730c>t p.r244x 1 (b, f) 5 c.814_815delct p.l272vfsx (g) 5 c.830t>c p.l277p (d) 5 c.835g>t p.e279x 1 (b) 5 c.868c>g p.h290d (b) 5 c.870c>g p.h290q (e) 5 c.881g>a p.w294x (f) 5 c.892g>t p.c301f (b) 6 c.919t>c p.c307r 1 this study 6 c.932g>a p.r311q 1 (a) (a) Regal et al., 2010 (d) Steinberg et al., 2009 (g) Okumoto et al., 1998a (b) Steinberg et al., 2004 (e) Warren et al., 1998 (c) Warren et al., 2000 (f) Krause et al.,

45 Genetic Classification and Mutational Spectrum of ZSS Mutations in the PEX12 gene are the third common cause of a ZSS disorder: 9% of our patient group has a mutation in PEX12. PEX12 is one of the RING finger proteins; it contains two transmembrane domains and a C-terminal zinc-binding motif. All 34 identified PEX12 mutations are listed in table 7, including 8 that have not been reported before. The ps320f mutation (c.959c>t) is the most common PEX12 mutation and is located in the zinc-binding domain. Patients homozygous Table 7. Mutations in the PEX12 gene. Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygous Heterozygous Reference 1 c.16dupg p.a6gfsx11 1 this study 1 c.26_27delca p.t9sfsx7 (a) 1 c.102a>t p.r34s 1 (b) 1 c.126+1g>t (a) 2 c.202_204delctt p.l68del (a) 2 c.220t>g p.y74d 1 this study 2 c.260_261insaa p.y87x (c) 2 c.268_271delaaga p.k90efsx3 (a) 2 c.273a>t p.r91s 1 (d) 2 c.308_309inst p.l103ffsx3 1 (d) 2 c.378_380delttc p.l126del 1 this study 2 c.445_454deltcttcccgct p.s149gfsx15 1 this study 2 c.460c>t p.r154x 1 this study 2 c.478_479delca p.y156x 1 this study 2 c.531_533delaca p.q178del (e) 2 c.533_535delaac p.q178del 1 1 (c) 2 c.538c>t p.r180x 2 (f, g) 2 c.541_542inst p.y181lfsx37 (c) 2 c.604c>t p.r202x 3 (d) 2 c.625c>t p.q209x 2 (d) i2 c.681-2a>c - (c) 3 c.684_687deltagt p.s222rfsx3 (a) 3 c.691a>t p.k231x (h) 3 c.733_734dupgcct p.l245cfsx19 1 (c, f) 3 c.744_745inst p.t249yfsx14 1 (a) 3 c.775c>t p.q259x 1 this study 3 c.875_876delct p.s292x 1 (g) 3 c.887_888deltc p.l296fs3007x 1 1 (c, d, f) 3 c.887delt p.l296pfsx5 (c) 3 c.888_889delct p.l297tfsx (a, c) 3 c.920g>a p.c307y 1 this study 3 c.949c>t p.l317f 1 (d) 3 c.959c>t p.s320f 13 2 (c) 3 c.1047_1049delaca p.q349del 2 (c) 2 (a) Chang and Gould, 1998 (d) Gootjes et al., 2004c (g) Okumoto et al., 1998b (b) Zeharia et al., 2007 (e) Yik et al., 2009 (h) Okumoto and Fujiki, 1997 (c) Steinberg et al., 2004 (f) Chang et al.,

46 Chapter 2 for this mutation presented an atypical biochemical phenotype in skin fibroblasts: the peroxisomal parameters are mostly normal to slightly abnormal. The mutation causes a temperature-sensitive phenotype: when cultured at 30 C, all cells contain catalase import competent peroxisomes, when cultured at 37 C only, 70% of the cells contain catalase positive peroxisomes and when cultured at 40 C none of the cells contain catalase positive peroxisomes (Gootjes et al., 2004a). 2 Table 8. Mutations in the PEX13 gene. Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygous Heterozygous Reference i1 c.92+2t>g - 1 this study 2 c.107_120del p.g36dfsx61 (a) 2 c.439_441delatg p.m147del 1 this study 2 c.676c>t p.r226x 2 this study 2 c.702g>a p.w234x 1 (b) 4 c.937t>g p.w313g (c) 4 c.970g>t p.g324x (b) 4 c.977t>c p.i326t 1 (b, d) 4 c.980c>g p.p327r 1 this study 1-4 c.1_ (a) 1 Deletion of 147,308 bp that included the PEX13 gene and 70,094 bp upstream and 45,692 bp downstream of the PEX13 gene (a) Al-Dirbashi et al., 2009 (c) Krause et al., 2006 (b) Shimozawa et al., 1999a (d) Liu et al., 1999 PEX13 is a peroxisomal integral membrane protein with two putative membrane domains. The C-terminus contains an SH3 domain, which binds to pentapeptide repeats 2-4 of PEX5. The N-terminal part of yeast PEX13 interacts with PEX7. Table 8 lists ten different PEX13 mutations of which six have been reported before. The I326T mutation causes a temperature-sensitive biochemical phenotype in fibroblasts: peroxisomes still import residual levels of PTS1 and PTS2 proteins at 37 C but not at elevated temperature. (Liu et al., 1999; Shimozawa et al., 1999b). Table 9. Mutations in the PEX14 gene. Mutaties Exon Nucleotide Amino acid Number of patients identified in this study Homozygous Heterozygous Reference 3 c.85-?_170+?del p.[ile29_lys56del;gly57glyfsx2] (a) 7 c.553c>t p.gln185ter (b) 7 c.585+1g>t r.585+1_37ins 1 this study (a) Huybrechts et al., 2008 (b) Shimozawa et al.,

47 Genetic Classification and Mutational Spectrum of ZSS The PEX14 gene encodes a peroxisomal membrane protein that was initially identified as a peroxisomal docking factor for the PTS1 receptor PEX5 via binding to the amino-terminal WxxxF/Y motifs of PEX5. More recently, it was proposed that PEX14 is also the site from which PEX5 leaves the peroxisomal compartment (Azevedo and Schliebs, 2006). A mutation in PEX14 is the least common cause of ZZS disorder. Three different mutations have now been identified in the PEX14 gene of which 1 has not been reported previously (Table 9). The PEX16 (MIM603360) gene encodes an integral peroxisomal membrane protein involved in peroxisomal membrane assembly. Two different groups of PEX16- defective patients have been reported; patients with a severe clinical presentation of whom the fibroblasts displayed a defect in import of peroxisomal matrix and membrane proteins, resulting in a total absence of peroxisomal remnants (Honsho et al., 1998; Shimozawa et al., 2002) and, very recently, several patients with a relatively mild clinical phenotype of the fibroblasts showed enlarged, import-competent peroxisomes (Ebberink et al, 2010b). In total eight different mutations have been identified in the PEX16 gene of which one has not been reported previously (Table 10). 2 Table 10. Mutations in the PEX16 gene. Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygous Heterozygous Reference i5 c insg - 1 this study 6 c.526c>t p.r176x (a, b) 8 c.753_755deltgt p.v252del 1 (c) 9 c.865c>a p.p289t 1 (c) i11 c _ (c) i10 c.952+2t>c R298fsX38 2 (d) 11 c.984delg p.i330sfsx27 1 (c) 11 c.992a>g p.y331c 1 (c) (a) Honsho et al., 1998 (c) Ebberink et al., 2010b (d) Shimozawa et al., 2002 (b) South and Gould, 1999 The PEX19 (MIM600279) gene encodes a mainly cytosolic protein that binds many peroxisomal membrane proteins, suggesting that PEX19 functions as a peroxisomal receptor for PMP (Sacksteder et al., 2000). The patients with a defect in PEX19 presented with a severe ZS phenotype and the fibroblasts displayed the absence of peroxisomal ghosts (Matsuzono et al., 1999). Table 11 lists three different PEX19 mutations of which two have not been reported before. PEX26 (MIM608666) is a membrane protein with one putative membrane-spanning domain. Human PEX26 is the ortholoque of yeast PEX15. Some of the reported mutations were found to lead to temperature-sensitive biochemical phenotype (Matsumoto et al., 2003a; Matsumoto et al., 2003b). Combined with previously reported PEX26 mutations, 22 different mutations have now been identified in the PEX26 gene of which three have not been reported previously (Table 12). 45

48 Chapter 2 Table 11. Mutations in the PEX19 gene. 2 Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygous Heterozygous Reference 3 c.320dela p.k320fsx13 1 this study 6 c.739c>t p.q257x 1 this study 6 c.763_764insa p.m255nfsx24 1 (a) (a) Matsuzono et al., 1999 Table 12. Mutations in the PEX26 gene. Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygous Heterozygous Reference 2 c.2t>c disruption startcodon (a, b) 2 c.34_35insc p.l12pfsx103 2 (a) 2 c.37_38delag p.r13gfsx164 (c) 2 c.73_79delgtgcgcg p.v25rfsx55 1 this study 2 c.131t>c p.l44p 1 (b) 2 c.134t>c p.l45p (a, b) 2 c.185g>a p.w62x 1 this study 2 c.192_216del p.s64rfsx10 (c) i2 c.230+1g>t p.t77fsx139 (b) 3 c.254_255inst p.l85lfsx93 (a, b) 3 c.265g>a p.g89r (a) 3 c.292c>t p.r98w 10 2 (a, b, c) 3 c.296g>a p.w99x (c) 3 c.315g>a p.w105x 1 this study 3 c.350c>t p.p117l (b) 3 c.353c>g p.p118r (c) i3 c.371+2t>c - (c) 4 c.426_548dup122bpinst p.a143_v182dup (b) +G183V 4 c.574c>t p.r192x 1 (c) i4 c.667+2t>c - (c) 5 r.668_814 p.g223_p271del (a) 6 c.862delc p.r288afsx366 (b) (a) Matsumoto et al., 2003b (b) Weller et al., 2005 (c) Steinberg et al., 2004 Conclusions In summary, we developed an efficient genetic complementation assay, which allows for rapid identification of the defective PEX gene in fibroblast cell lines of ZSS patients. This assay has been included in our diagnostic work up of patients diagnosed with a ZSS disorder, followed by sequence analysis of the defective PEX gene. After analysis of 613 independent cell lines, we did not detect cell lines with a novel defective PEX gene suggesting that only mutations in one of the currently known PEX genes leads to complete peroxisome deficiency and a Zellweger Syndrome-like presentation. 46

49 Genetic Classification and Mutational Spectrum of ZSS Acknowledgements We thank to Dr. S.J. Gould for the gift of PEX pcdna3 plasmids, J. Haasjes, W. Oostheim, M, de Gijsel and R.A. Jibodh for their assistance in different parts of this study and Dr. S. Ferdinandusse for critical reading of the manuscript. This study was supported by a grant from the Prinses Beatrix Fonds (MAR 03_0216) and by the FP6 European Union Project peroxisomes (LSHG-CT ). We gratefully acknowledge all doctors and medical specialists who referred patient material to our laboratory. 2 References Al-Dirbashi OY, Shaheen R, Al-Sayed M, Al-Dosari M, Makhseed N, Abu SL, Santa T, Meyer BF, Shimozawa N, Alkuraya FS Zellweger syndrome caused by PEX13 deficiency: report of two novel mutations. Am J Med Genet A 149A: Azevedo JE, Schliebs W Pex14p, more than just a docking protein. Biochim Biophys Acta 1763: Braverman N, Chen L, Lin P, Obie C, Steel G, Douglas P, Chakraborty PK, Clarke JT, Boneh A, Moser A, Moser H, Valle D Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype. Hum Mutat 20: Brul S, Westerveld A, Strijland A, Wanders RJ, Schram AW, Heymans HS, Schutgens RB, van den Bosch H, Tager JM Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis. J Clin Invest 81: Chang CC, Gould SJ Phenotype-genotype relationships in complementation group 3 of the peroxisome-biogenesis disorders. Am J Hum Genet 63: Chang CC, Warren DS, Sacksteder KA, Gould SJ PEX12 interacts with PEX5 and PEX10 and acts downstream of receptor docking in peroxisomal matrix protein import. J Cell Biol 147: Collins CS, Gould SJ Identification of a common PEX1 mutation in Zellweger syndrome. Hum Mutat 14: Dodt G, Braverman N, Wong C, Moser A, Moser HW, Watkins P, Valle D, Gould SJ Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nat Genet 9: Dodt G, Warren D, Becker E, Rehling P, Gould SJ Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p. J Biol Chem 276: Dursun A, Gucer S, Ebberink MS, Yigit S, Wanders RJ, Waterham HR Zellweger syndrome with unusual findings: non-immune hydrops fetalis, dermal erythropoiesis and hypoplastic toe nails. J Inherit Metab Dis. Ebberink MS, Koster J, Wanders RJ, Waterham HR. 2010a. Spectrum of PEX6 mutations in Zellweger syndrome spectrum patients. Hum Mutat 31:E Ebberink MS, Mooyer PA, Koster J, Dekker CJ, Eyskens FJ, Dionisi-Vici C, Clayton PT, Barth PG, Wanders RJ, Waterham HR Genotype-phenotype correlation in PEX5-deficient peroxisome biogenesis defective cell lines. Hum Mutat 30: Ebberink MS, Csanyi B, Chong WK, Denis S, Sharp P, Mooijer PAW, Dekker CJM, Spooner C, Ngu LH, De Sousa C, Wanders RJA, Fietz MJ, Clayton PT, Waterham HR, Ferdinandusse S. 2010b. Identification of an unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene. J Med Genet. (accepted) Gartner J, Preuss N, Brosius U, Biermanns M Mutations in PEX1 in peroxisome biogenesis disorders: G843D and a mild clinical phenotype. J Inherit Metab Dis 22: Ghaedi K, Honsho M, Shimozawa N, Suzuki Y, Kondo N, Fujiki Y PEX3 is the causal gene responsible for peroxisome membrane assembly-defective Zellweger syndrome of complementation group G. Am J Hum Genet 67: Gootjes J, Mandel H, Mooijer PA, Roels F, Waterham HR, Wanders RJ Resolution of the molecular 47

50 Chapter 2 2 defect in a patient with peroxisomal mosaicism in the liver. Adv Exp Med Biol 544: Gootjes J, Schmohl F, Waterham HR, Wanders RJ. 2004a. Novel mutations in the PEX12 gene of patients with a peroxisome biogenesis disorder. Eur J Hum Genet 12: Gootjes J, Skovby F, Christensen E, Wanders RJ, Ferdinandusse S. 2004b. Reinvestigation of trihydroxycholestanoic acidemia reveals a peroxisome biogenesis disorder. Neurology 62: Gootjes J, Elpeleg O, Eyskens F, Mandel H, Mitanchez D, Shimozawa N, Suzuki Y, Waterham HR, Wanders RJ. 2004c. Novel mutations in the PEX2 gene of four unrelated patients with a peroxisome biogenesis disorder. Pediatr Res 55: Gootjes J, Schmohl F, Mooijer PA, Dekker C, Mandel H, Topcu M, Huemer M, Von Schutz M, Marquardt T, Smeitink JA, Waterham HR, Wanders RJ. 2004d. Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism. Hum Mutat 24: Grou CP, Carvalho AF, Pinto MP, Wiese S, Piechura H, Meyer HE, Warscheid B, Sa-Miranda C, Azevedo JE Members of the E2D (UbcH5) Family Mediate the Ubiquitination of the Conserved Cysteine of Pex5p, the Peroxisomal Import Receptor. J Biol Chem 283: Honsho M, Tamura S, Shimozawa N, Suzuki Y, Kondo N, Fujiki Y Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D. Am J Hum Genet 63: Huybrechts SJ, Van Veldhoven PP, Hoffman I, Zeevaert R, de Vos R, Demaerel P, Brams M, Jaeken J, Fransen M, Cassiman D Identification of a novel PEX14 mutation in Zellweger syndrome. J Med Genet 45: Imamura A, Tamura S, Shimozawa N, Suzuki Y, Zhang Z, Tsukamoto T, Orii T, Kondo N, Osumi T, Fujiki Y. 1998a. Temperature-sensitive mutation in PEX1 moderates the phenotypes of peroxisome deficiency disorders. Hum Mol Genet 7: Imamura A, Tsukamoto T, Shimozawa N, Suzuki Y, Zhang Z, Imanaka T, Fujiki Y, Orii T, Kondo N, Osumi T. 1998b. Temperature-sensitive phenotypes of peroxisome-assembly processes represent the milder forms of human peroxisome-biogenesis disorders. Am J Hum Genet 62: Krause C, Rosewich H, Thanos M, Gartner J Identification of novel mutations in PEX2, PEX6, PEX10, PEX12, and PEX13 in Zellweger spectrum patients. Hum Mutat 27:1157. Krause C, Rosewich H, Gartner J Rational diagnostic strategy for Zellweger syndrome spectrum patients. Eur J Hum Genet. Liu Y, Bjorkman J, Urquhart A, Wanders RJ, Crane DI, Gould SJ PEX13 is mutated in complementation group 13 of the peroxisome-biogenesis disorders. Am J Hum Genet 65: Matsumoto N, Tamura S, Fujiki Y. 2003a. The pathogenic peroxin Pex26p recruits the Pex1p-Pex6p AAA ATPase complexes to peroxisomes. Nat Cell Biol 5: Matsumoto N, Tamura S, Furuki S, Miyata N, Moser A, Shimozawa N, Moser HW, Suzuki Y, Kondo N, Fujiki Y. 2003b. Mutations in novel peroxin gene PEX26 that cause peroxisome-biogenesis disorders of complementation group 8 provide a genotype-phenotype correlation. Am J Hum Genet 73: Matsuzono Y, Kinoshita N, Tamura S, Shimozawa N, Hamasaki M, Ghaedi K, Wanders RJ, Suzuki Y, Kondo N, Fujiki Y Human PEX19: cdna cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly. Proc Natl Acad Sci U S A 96: Maxwell MA, Allen T, Solly PB, Svingen T, Paton BC, Crane DI Novel PEX1 mutations and genotype-phenotype correlations in Australasian peroxisome biogenesis disorder patients. Hum Mutat 20: Maxwell MA, Leane PB, Paton BC, Crane DI Novel PEX1 coding mutations and 5 UTR regulatory polymorphisms. Hum Mutat 26:279. Motley AM, Brites P, Gerez L, Hogenhout E, Haasjes J, Benne R, Tabak HF, Wanders RJ, Waterham HR Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1. Am J Hum Genet 70: Muntau AC, Mayerhofer PU, Paton BC, Kammerer S, Roscher AA Defective peroxisome membrane synthesis due to mutations in human PEX3 causes Zellweger syndrome, complementation group G. Am J Hum Genet 67: Okumoto K, Fujiki Y PEX12 encodes an integral membrane protein of peroxisomes. Nat Genet 17:

51 Genetic Classification and Mutational Spectrum of ZSS Okumoto K, Itoh R, Shimozawa N, Suzuki Y, Tamura S, Kondo N, Fujiki Y. 1998a. Mutations in PEX10 is the cause of Zellweger peroxisome deficiency syndrome of complementation group B. Hum Mol Genet 7: Okumoto K, Shimozawa N, Kawai A, Tamura S, Tsukamoto T, Osumi T, Moser H, Wanders RJ, Suzuki Y, Kondo N, Fujiki Y. 1998b. PEX12, the pathogenic gene of group III Zellweger syndrome: cdna cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p. Mol Cell Biol 18: Platta HW, Erdmann R The peroxisomal protein import machinery. FEBS Lett 581: Platta HW, El MF, Baumer BE, Schlee D, Girzalsky W, Erdmann R Pex2 and pex12 function as protein-ubiquitin ligases in peroxisomal protein import. Mol Cell Biol 29: Portsteffen H, Beyer A, Becker E, Epplen C, Pawlak A, Kunau WH, Dodt G Human PEX1 is mutated in complementation group 1 of the peroxisome biogenesis disorders. Nat Genet 17: Preuss N, Brosius U, Biermanns M, Muntau AC, Conzelmann E, Gartner J PEX1 mutations in complementation group 1 of Zellweger spectrum patients correlate with severity of disease. Pediatr Res 51: Régal L, Ebberink MS, Goemans N, Wanders RJA, De Meirleir L, Jaeken J, Schrooten M, Van Coster R, Waterham HR Mutations in PEX10 are a cause of autosomal recessive ataxia. Ann Neurol. (accepted) Reuber BE, Germain-Lee E, Collins CS, Morrell JC, Ameritunga R, Moser HW, Valle D, Gould SJ Mutations in PEX1 are the most common cause of peroxisome biogenesis disorders. Nat Genet 17: Rosewich H, Ohlenbusch A, Gärtner J Genetic and clinical aspects of Zellweger spectrum patients with PEX1 mutations. J Med Genet Sep;42(9):e58. Sacksteder KA, Jones JM, South ST, Li X, Liu Y, Gould SJ PEX19 binds multiple peroxisomal membrane proteins, is predominantly cytoplasmic, and is required for peroxisome membrane synthesis. J Cell Biol 148: Saidowsky J, Dodt G, Kirchberg K, Wegner A, Nastainczyk W, Kunau WH, Schliebs W The diaromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14. J Biol Chem 276: Shimozawa N, Suzuki Y, Orii T, Moser A, Moser HW, Wanders RJ Standardization of complementation grouping of peroxisome-deficient disorders and the second Zellweger patient with peroxisomal assembly factor-1 (PAF-1) defect. Am J Hum Genet 52: Shimozawa N, Suzuki Y, Tomatsu S, Nakamura H, Kono T, Takada H, Tsukamoto T, Fujiki Y, Orii T, Kondo N A novel mutation, R125X in peroxisome assembly factor-1 responsible for Zellweger syndrome. Hum Mutat Suppl 1:S134-S136. Shimozawa N, Zhang Z, Suzuki Y, Imamura A, Tsukamoto T, Osumi T, Fujiki Y, Orii T, Barth PG, Wanders RJ, Kondo N. 1999a. Functional heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5- defective patients. Biochem Biophys Res Commun 262: Shimozawa N, Suzuki Y, Zhang Z, Imamura A, Toyama R, Mukai S, Fujiki Y, Tsukamoto T, Osumi T, Orii T, Wanders RJ, Kondo N. 1999b. Nonsense and temperature-sensitive mutations in PEX13 are the cause of complementation group H of peroxisome biogenesis disorders. Hum Mol Genet 8: Shimozawa N, Suzuki Y, Zhang Z, Imamura A, Ghaedi K, Fujiki Y, Kondo N. 2000a. Identification of PEX3 as the gene mutated in a Zellweger syndrome patient lacking peroxisomal remnant structures. Hum Mol Genet 9: Shimozawa N, Zhang Z, Imamura A, Suzuki Y, Fujiki Y, Tsukamoto T, Osumi T, Aubourg P, Wanders RJ, Kondo N. 2000b. Molecular mechanism of detectable catalase-containing particles, peroxisomes, in fibroblasts from a PEX2-defective patient. Biochem Biophys Res Commun 268: Shimozawa N, Nagase T, Takemoto Y, Suzuki Y, Fujiki Y, Wanders RJ, Kondo N A novel aberrant splicing mutation of the PEX16 gene in two patients with Zellweger syndrome. Biochem Biophys Res Commun 292: Shimozawa N, Tsukamoto T, Nagase T, Takemoto Y, Koyama N, Suzuki Y, Komori M, Osumi T, Jeannette G, Wanders RJ, Kondo N Identification of a new complementation group of the peroxisome biogenesis disorders and PEX14 as the mutated gene. Hum Mutat 23: South ST, Gould SJ Peroxisome synthesis in the absence of preexisting peroxisomes. J Cell Biol 144: South ST, Sacksteder KA, Li X, Liu Y, Gould SJ Inhibitors of COPI and COPII do not block PEX3-2 49

52 Chapter 2 2 mediated peroxisome synthesis. J Cell Biol 149: Steinberg S, Chen L, Wei L, Moser A, Moser H, Cutting G, Braverman N The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum. Mol Genet Metab 83: Steinberg SJ, Dodt G, Raymond GV, Braverman NE, Moser AB, Moser HW Peroxisome biogenesis disorders. Biochim Biophys Acta 1763: Steinberg SJ, Snowden A, Braverman NE, Chen L, Watkins PA, Clayton PT, Setchell KD, Heubi JE, Raymond GV, Moser AB, Moser HW A PEX10 defect in a patient with no detectable defect in peroxisome assembly or metabolism in cultured fibroblasts. J Inherit Metab Dis 32: Tamura S, Okumoto K, Toyama R, Shimozawa N, Tsukamoto T, Suzuki Y, Osumi T, Kondo N, Fujiki Y Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I. Proc Natl Acad Sci U S A 95: Tamura S, Matsumoto N, Imamura A, Shimozawa N, Suzuki Y, Kondo N, Fujiki Y Phenotypegenotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by Pex1p-Pex6p interaction. Biochem J 357: Walter C, Gootjes J, Mooijer PA, Portsteffen H, Klein C, Waterham HR, Barth PG, Epplen JT, Kunau WH, Wanders RJ, Dodt G Disorders of peroxisome biogenesis due to mutations in PEX1: phenotypes and PEX1 protein levels. Am J Hum Genet 69: Wanders RJ, Waterham HR Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders. Clin Genet 67: Wanders RJ, Waterham HR Biochemistry of mammalian peroxisomes revisited. Annu Rev Biochem 75: Warren DS, Morrell JC, Moser HW, Valle D, Gould SJ Identification of PEX10, the gene defective in complementation group 7 of the peroxisome-biogenesis disorders. Am J Hum Genet 63: Warren DS, Wolfe BD, Gould SJ Phenotype-genotype relationships in PEX10-deficient peroxisome biogenesis disorder patients. Hum Mutat 15: Waterham HR, Koster J, van Roermund CW, Mooyer PA, Wanders RJ, Leonard JV A lethal defect of mitochondrial and peroxisomal fission. N Engl J Med 356: Weller S, Gould SJ, Valle D Peroxisome biogenesis disorders. Annu Rev Genomics Hum Genet 4: Weller S, Cajigas I, Morrell J, Obie C, Steel G, Gould SJ, Valle D Alternative splicing suggests extended function of PEX26 in peroxisome biogenesis. Am J Hum Genet 76: Yik WY, Steinberg SJ, Moser AB, Moser HW, Hacia JG Identification of novel mutations and sequence variation in the Zellweger syndrome spectrum of peroxisome biogenesis disorders. Hum Mutat 30:E467-E480. Zeharia A, Ebberink MS, Wanders RJ, Waterham HR, Gutman A, Nissenkorn A, Korman SH A novel PEX12 mutation identified as the cause of a peroxisomal biogenesis disorder with mild clinical phenotype, mild biochemical abnormalities in fibroblasts and a mosaic catalase immunofluorescence pattern, even at 40 degrees C. J Hum Genet 52:

53 Chapter 3 Genotype-Phenotype Correlation in PEX5-Deficient Peroxisome Biogenesis Defective Cell Lines Merel S. Ebberink 1, Petra A.W. Mooyer 1, Janet Koster 1, Conny J.M Dekker 1, François J.M. Eyskens 2, Carlo Dionisi-Vici 3, Peter T. Clayton 4, Peter G. Barth 5, Ronald J.A. Wanders 1,5 and Hans R. Waterham 1,5 Academic Medical Centre, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry 1, Department of Paediatrics 5 /Emma Children s Hospital, Amsterdam, The Netherlands. 2 The University Hospital of Antwerp, Metabolic Unit, Antwerp, Belgium. 3 Bambino Gesù Children s Hospital, Division of Metabolism, Rome, Italy. 4 Biochemistry Research Group, UCL Institute of Child Health, London, United Kingdom. Human Mutation (2009) 30(1):93-98

54 Chapter 3 Abstract 3 Proteins destined for the peroxisomal matrix are targeted by virtue of a peroxisomal targeting sequence type 1 (PTS1) or type 2 (PTS2). In humans, targeting of either class of proteins relies on a cytosolic receptor protein encoded by the PEX5 gene. Alternative splicing of PEX5 results in two protein variants, PEX5S and PEX5L. PEX5S is exclusively involved in PTS1 protein import, whereas PEX5L mediates the import of both PTS1 and PTS2 proteins. Genetic complementation testing with over 500 different fibroblast cell lines from patients diagnosed with a peroxisome biogenesis disorder identified eleven cell lines with a defect in PEX5. The aim of this study was to characterize these cell lines at a biochemical and genetic level. To this end, the cultured fibroblasts were analyzed for very long chain fatty acid concentrations, peroxisomal β-and α-oxidation, Dihydroxyacetonephosphate Acyltransferase (DHAPAT) activity, peroxisomal thiolase and catalase immunofluorescence. Mutation analysis of the PEX5 gene revealed eleven different mutations, eight of which are novel. PTS1- and PTS2- protein import capacity was assessed by transfection of the cells with Green Fluorescent Protein (GFP) tagged with either PTS1 or PTS2. Six cell lines showed a defect in both PTS1 and PTS2 protein import, whereas four cell lines only showed a defect in PTS1 protein import. The location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in the cell lines. 52

55 Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines Introduction Human peroxisomes play an important role in various essential metabolic pathways, among which the biosynthesis of ether phospholipids and the alpha- and beta-oxidation of fatty acids (Wanders and Waterham, 2006). Consequently, defects in genes encoding peroxisomal proteins can lead to a variety of different peroxisomal disorders that can be categorized in two main groups, including the peroxisome biogenesis disorders (PBDs) and the single peroxisomal enzyme deficiencies (Wanders and Waterham, 2006; Weller et al., 2003). The first group originally comprised of three defined phenotypes, including the Cerebro-Hepato-Renal Syndrome of Zellweger (i.e. Zellweger Syndrome; ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) with decreasing clinical and biochemical severity. Currently, however, they are referred to as the Zellweger Syndrome Spectrum (ZSS) based on the recognition that the different phenotypes can be caused by mutations in different genes, as well as different mutations within the same gene, and the fact that the three phenotypes show considerable clinical and biochemical overlap (Shimozawa et al., 1999; Steinberg et al., 2006). Peroxisomal matrix proteins are encoded by nuclear genes, synthesized on free cytosolic ribosomes and imported post-translationally into the peroxisomes (Lazarow and Fujiki, 1985). In humans, currently 13 different proteins, called peroxins, have been shown to be involved in specific stages of this import process (Platta and Erdmann, 2007; Figure 1). This involvement also follows from the fact that mutations in either of 12 of the PEX genes coding for these peroxins were found to result in a PBD of the (ZSS). 3 Figure 1. Model for peroxisomal matrix protein import in mammalian cells. PEX5S is exclusively involved in peroxisomal PTS1 protein import, whereas PEX5L mediates both PTS1 and PTS2 protein import. Peroxisomal protein import can be divided in four stages: 1) Binding of peroxisomal matrix proteins to their receptor. 2) Docking of the receptor-ligand complex to the peroxisome. 3) Translocation of the receptor-ligand complex and release of the ligand into the matrix. 4) Receptor recycling into the cytosol or receptor degradation via the proteasome (for details, see Platta et al., 2007). 53

56 Chapter 3 3 The targeting of peroxisomal matrix proteins to peroxisomes is mediated by peroxisomal targeting sequences (PTS), which are recognized by specific cytosolic receptor proteins. The majority of peroxisomal proteins contain a carboxy-terminal tripeptide with a conserved consensus sequence, S/A/C-K/R/H-L/M, called PTS1, which is recognized by the cytosolic receptor protein PEX5 (MIM ). In addition, few proteins have an amino-terminal PTS2 with consensus R/K-L/V-X5- H/Q-L/A, which is recognized by the cytosolic receptor protein PEX7 (MIM ; (Dodt et al., 2001). Accordingly, defects in PEX5 in principle result in an inability to import PTS1 proteins leading to a generalized peroxisome biogenesis defect, whereas a defect in PEX7 only affects the import of a small subset of peroxisomal proteins leading to a different clinical presentation, i.e. Rhizomelic Chondrodysplasia Punctata (RCDP; Motley et al., 2002). Human PEX5 is a 67-kD protein with seven di-aromatic pentapeptide repeats (WxxxF/Y) in its amino-terminal half and seven tetrapeptide repeats (TPRs) in its carboxy-terminal half (Gatto et al., 2000). The TPR-containing carboxy-terminal half of PEX5 has been shown to mediate the interaction with the PTS1 sequence, whereas the WxxxF/Y motifs in the amino-terminal half of PEX5 appear essential for docking to the peroxisomal membrane and for binding to either PEX13 (MIM ) or PEX14 (MIM (Saidowsky et al., 2001; Weller et al., 2003). In humans, two functional protein variants of PEX5 are produced as a result of alternative splicing of the PEX5 mrna. The longest variant, PEX5L, contains an additional 111bp encoding 37 amino acids, due to alternative splicing of exon 7 (Dodt et al., 2001). The shorter protein, PEX5S, has been reported to be exclusively involved in peroxisomal PTS1 protein import, whereas PEX5L mediates both PTS1 and PTS2 protein import (Figure1). In fact, docking of the PEX7-PTS2 protein complex to the PEX13 and PEX14 proteins of the peroxisomal import machinery can only occur through physical interaction with PEX5L. The PEX5L region involved in this interaction includes part of the PEX5Lspecific insertion (Braverman et al., 1998). In the past two years, we assigned over 500 cell lines from patients diagnosed with ZS to different genetic complementation groups by means of functional complementation assays (manuscript in preparation, but see also chapter 2). Among these cell lines we identified 11 cell lines with a defect in PEX5. We here report the biochemical and genetic characterization of these cell lines and show that the location of the mutations within specific domains of the PEX5 protein correlates rather well with the specific peroxisomal import defect observed in these cell lines. Materials and Methods Patient cell lines For this study we used primary skin fibroblasts from patients who, based on clinical and biochemical characteristics, were diagnosed with ZS and which were sent to our laboratory for diagnostic workup. The cells were cultured in DMEM medium with 4.5g/L glucose and L- glutamine (BioWhittaker) or HAM F-10 medium with L-glutamine and Hepes 25mM (Gibco, Invitrogen), supplemented with 10% fetal bovine serum (FBS, BioWhittaker), 100 U/ml penicillin, 100µg/ml streptomycin and 54

57 Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines 25 mm Hepes buffer (DMEM media only) in a humidified atmosphere of 5% CO 2 and at 37 C. DMEM medium is used for the transfection experiments, HAM F-10 medium for the biochemical analysis and the complementation assay via polyethylene glycolmediated (PEG) fusion. The cells were determined to be defective in the PEX5 gene by means of genetic complementation analysis, involving either a previously described PEG fusion method (Brul et al., 1988) or our recently developed PEX cdna transfection method (manuscript in preparation, but see also chapter 2). In accordance to the institutional guidelines and the Dutch Code of Conduct, identifiable clinical and personal data from the patients were not available for this study. Biochemical analysis DHAPAT activity (Ofman and Wanders, 1994), concentrations of very long chain fatty acids (VLCFAs; (Vreken et al., 1998), β-oxidation of C26:0, C16:0 and pristanic acid (Wanders et al., 1995b), and α-oxidation of phytanic acid (Wanders and van Roermund, 1993) were measured in the cultured fibroblasts as previously described. Catalase immunofluorescence (van Grunsven et al., 1999) and immunoblot analysis using an antibody against peroxisomal thiolase 1 (Wanders et al., 1995a) were performed as described before. 3 Assessment of PTS1 and PTS2 protein import Peroxisomal import of PTS1 and PTS2 proteins was assessed by transfection of cultured fibroblasts with GFP fused to either a carboxy-terminal PTS1 (egfp-pts1) or an amino-terminal PTS2 signal (PTS2-GFP) using the AMAXA nucleofector technology (Amaxa, Cologne, Germany). Two days after transfection, the cells were examined for the subcellular localization of the GFP protein using fluorescence microscopy with a nm filter. Table 1. Primer sets used for PEX5 mutation analysis. amplicon 5 primer (forward) 3 primer (reverse) exon 1 and 2 [-21M13]- ACg ggc AgA gtt gtg gat g [M13-Rev]- ATT gaa ATA Cgg gtg AAC TAA g exon 3 and 4 [-21M13]- AgC CTA Tgg gtt CAT TTC ATC [M13-Rev]- AgA ATT CTg TCC CAT AgA AgC exon 5 [-21M13]- TCA gtt gaa TAT ggg CAT CTC [M13-Rev]- TgT CCA TAC TCC TTT CAC exon 6 [-21M13]- ACA gga ACT gtc ATT gtc ATg [M13-Rev]- CAg gaa CgA AgA gac CTA Ag exon 7 and 8 [-21M13]- Tgg AAg TCC TTT CCC AAg Tg [M13-Rev]- TCC Agg TCC ACT ATg AAA TAC exon 9 [-21M13]- TgA AAT TCA AgA ACT gct gcc [M13-Rev]- gaa gga AgT TCT gga ACC Tg exon 10 and 11 [-21M13]- CTg CCT gct ggt TgT CAT C [M13-Rev]- AAg ACA Agg ATC CAg gtc Tg exon 12 and 13 [-21M13]- AgC TTg gct Tgg ATC CCA g [M13-Rev]- ACA ggc ATg CAC CAT CAA AC exon 14 and 15 [-21M13]- CCT gga gta ATg TgC AgA g [M13-Rev]- gta CCg CTT ATg gtc ATC Ag c.-11_c.657 [-21M13]- TGG CGG TCA CCA TGG CAA TG [M13-Rev]- GGC ATC TGA TGT ACC CTC AG c.563_c.1226 [-21M13]- ATC ATC CTG AGG AGG ATC TG [M13-Rev]- CCT TCT TCA GCA GGT GTC AC c.1154_c.1840 [-21M13]- CCT GTG AAA TCC TAC GAG AC [M13-Rev]- ATC CCT CCA GGT GGA CAC TC All forward and reverse primers were tagged with a -21M13 (5- TGTAAAACGACGGCCAGT-3 ) sequence or M13rev (5 -CAGGAAACAGCTATGACC-3 ) sequence, respectively. 55

58 Chapter 3 3 Mutation analysis PEX5 mutation analysis was performed by sequencing all exons plus flanking intronic sequences of the PEX5 gene amplified by PCR from genomic DNA isolated from fibroblasts and using the primer sets shown in table 1. To determine the effect of certain mutations on post-transcriptional level, we also sequenced PEX5 cdnas prepared from mrna isolated from cultured fibroblasts. Primer sets for cdna analysis are also shown in table 1. Genomic DNA was isolated from skin fibroblasts using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). Total RNA was isolated from skin fibroblasts using Trizol (Invitrogen, Carlsbad, CA) extraction, after which cdna was prepared using a first strand cdna synthesis kit for RT-PCR (Roche, Mannheim, Germany). All forward and reverse primers were tagged with a -21M13 (5- TGTAAAACGACGGCCAGT-3 ) sequence or M13rev (5 -CAGGAAACAGCTATGACC-3 ) sequence, respectively. PCR fragments were sequenced in two directions using -21M13 and M13rev primers by means of BigDye Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA) and analyzed on an Applied Biosystems 377A automated DNA sequencer, following the manufacturer s protocol (Applied Biosystems, Foster City, CA, USA). The PEX5 sequences were compared to the reference sequence of PEX5L (GenBank accession number X84899) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. Results Biochemical Analysis After genetic complementation analysis of more than 500 different skin fibroblast cell lines from patients diagnosed with a peroxisomal biogenesis disorder, we identified 11 cell lines with a defect in the PEX5 gene. To determine the extent of peroxisomal dysfunction due to the PEX5 defect, we studied various peroxisomal parameters in the cultured skin fibroblasts, including DHAPAT activity, concentrations of C26:0 and C22:0 fatty acids, β-oxidation of C16:0, C26:0 and pristanic acid, phytanic acid α-oxidation, catalase immunofluorescence and thiolase immunoblot analysis (Table 2). This revealed a severe biochemical phenotype in ten cell lines (PEX5.1-PEX5.3, PEX5.5-PEX5.11) and a milder phenotype in cell line PEX5.4 Mutation analysis Sequence analysis of the coding region of the PEX5 gene revealed homozygous mutations in ten of the eleven cell lines (Table 3). In cell line PEX5.2, three different heterozygous mutations were detected in the PEX5 gene. Subsequent analysis of the PEX5 cdna showed that only one of these was expressed, indicating that the PEX5 transcription of the second PEX5 allele, containing two mutations, most probably results in an unstable mrna. We found four different missense mutations, one of which is the N526K mutation previously described in another PEX5-deficient patient (Braverman et al., 1998; Carvalho et al., 2007). This mutation is located in the carboxy-terminal TPRcontaining domain and thus predicted to block the binding of PTS1 proteins to the carboxy-terminal half of PEX5 (Figure 2). The S600W mutation was previously 56

59 Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines Table 2. Biochemical Parameters in the PEX5-Deficient Fibroblasts. immunoblot α-thiolase IF α-catalase DHAPAT nmol/2hr*mg α-oxidation pmol/hr*mg protein VLCFA µmol/g protein β-oxidation pmol/hr*mg protein Cell line C16:0 C26:0 Pristanic Acid C26:0 C26/C22 Phytanic acid 41kDa 44kDa Control a punctate + - ZS b difusse - + PEX diffuse - + PEX ND diffuse - + PEX ND 1.2 diffuse - + PEX diffuse - / + + PEX diffuse - + PEX ND diffuse - + PEX diffuse - + PEX diffuse - + PEX diffuse - + PEX diffuse - + PEX diffuse - + a Laboratory reference values for fibroblasts from controls. b Laboratory reference values for fibroblasts from patients with classical Zellweger syndrome. IF, immunofluorescence; ND, not done; punctate, peroxisomal catalase immunofluorescence pattern; diffuse, cytosolic catalase immunofluorescence pattern; +, present; -,absent. 3 57

60 Chapter 3 3 Table 3. Mutations in the PEX5 gene of cell lines PEX5.1 PEX5.11 and their consequences. Mutations Affected GFP-PTS1 PTS2-GFP cell line Nucleotide Amino Acid Exon protein domain import import PEX5.1 c.1578t>g pn526k 14 TPR6 - + PEX5.2 [c.1244a>g]+[c dupatcg; c.604g>c] p.n415s 1 11 TPR3 - + PEX5.3 c.1578t>g pn526k 14 TPR6 - + PEX5.4 c.1799c>g p.s600w 15 PTS1 binding site - + PEX5.5 c g>a inframe deletion 2 12 TPR PEX5.6 c.1669c>t p.r557w 14 TPR6 - - PEX5.7 c.826c>t p.r276x 7 PTS1 binding site - - PEX5.8 c.1090c>t p.q364x 10 TPR1 - - PEX5.9 c.1258c>t p.r420x 12 TPR3 - - PEX5.10 c.548_552-55delins238inv 3 incorrect splicing 4 5 repeat3 5 5 PEX5.11 c a>c incorrect splicing 6 14 TPR6 - - Reference sequence of PEX5L: GenBank accession number X Nucleotide numbering starting at the first adenine of the translation initiation codon ATG. 1 only allel 1 is expressed 2 inframe deletion of exon 12 leading to R394S and c del in PEX5 mrna 3 deletion of c.548_552-55, insertion of nucleotides _ inv (nucleotides of GenBank accession number NC_ range ) 4 deletion of exon 5, stop codon 33 amino acids after exon 4 5 cells did not survive transfection procedure 6 c del and c del observed in PEX5 mrna 58

61 Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines reported as S563W using a numbering system which was based on the shorter PEX5S transcript (Shimozawa et al., 1999). Ser600 is situated at the base of the 7C loop (Figure 2), which plays a central role in connecting the carboxy-terminal and amino-terminal TPR segments, to constitute the PTS1-binding site (Stanley et al., 2007). The other two missense mutations (N415S and R557W) have not been reported previously. The N415S mutation affects an asparagine located in the third TPR motif, whereas the R557W mutation is located in 6 th TPR motif. Because both TPR motifs are part of the binding site for PTS1 proteins (Figure 2), both mutations are expected to affect the binding of PTS1 proteins. Three different homozygous nonsense mutations were identified, which truncate the PEX5 upstream of the first TPR motif (R276X), within the first TPR motif (Q364X) or within the third TPR motif (R420X) respectively. PEX5 cdna semi-quantitative analysis revealed cdna levels similar to those observed in control cells indicating that all three nonsense mutations do not result in nonsense-mediated decay of the PEX5 mrnas. Cell line PEX5.5 was homozygous for a mutation located near the 3 Figure 2. Amino acid sequence of human PEX5L. The various domains within PEX5 are indicated above the sequence by different types of bars; the bars labelled with * indicate WxxxF/Y PEX14-binding motifs. The mutations found in the various PEX5 cell lines are marked in gray; the amino acids beneath the sequence indicate the resulting amino acid changes. A deletion is indicated by a gray bar under the sequence. GenBank accession number X

62 Chapter 3 3 Figure 3. Fibroblasts transfected with either GFP-PTS1 or PTS2-GFP. Shown are examples from transfection experiments to assess the degree of import deficiency for PTS1- and PTS2-targeted proteins. Shown are control cells (A and E), a PEX3-deficient cell line (B and F), the PEX5.2 cell line (C and G) and the PEX5.9 cell line (D and H). splice site junction of exon 12 and intron 12 (c g>a). Although the mutation does not affect an invariable nucleotide, it results in aberrant splicing as revealed by subsequent PEX5 cdna analysis, which showed skipping of exon 12. An unusual complex mutation was detected in cell line PEX5.10, where the last 6 nucleotides of exon 5 plus the first 55 nucleotides of intron 5 were substituted for a reverse complementary 45 nucleotide sequence normally located within intron 9 of the PEX5 gene. This indel mutation results in exon 5 skipping as shown by PEX5 cdna analysis. PEX5 cdna analysis of cell line PEX5.11 showed that the homozygous splice site mutation in this cell line (c a>c) has a dual effect including alternative splicing using a cryptic splice site at position 1584 (located within exon 14) or skipping of the entire exon 14. PTS1 and PTS2 protein import To assess the degree of import deficiency for PTS1- and PTS2-targeted proteins and to relate the deficiencies to the location of the various mutations in PEX5, we transfected the various cell lines with egfp-pts1 or PTS2-GFP reporter constructs (Figure 3). This showed that four cell lines have an isolated PTS1 protein import defect (patient PEX5.1-PEX5.4), whereas six cell lines showed a combined defect 60

63 Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines in both PTS1 and PTS2 protein import (patient PEX5.5-PEX5.9 and PEX5.11). Because the cells of PEX5.10 did not survive the transfection procedure despite several attempts, these studies could not be performed for this cell line. Discussion In the present study we characterized 11 different PEX5-deficient cell lines at the biochemical and genetic level. We identified 11 different mutations, including one small insertion, one indel, four missense, three nonsense and two splice site mutations. Eight of these mutations have not been described before, while previously only 3 patients with a PEX5 defect had been reported (Braverman et al., 1998; Dodt et al., 1995; Dodt et al., 2001; Shimozawa et al., 1999). We found that the location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in the cell lines. Except for cell line PEX5.4, the biochemical phenotypes of all cell lines were overall severe corresponding to the Zellweger Syndrome presentation. The biochemical defects observed in cell line PEX5.4 were less severe, including a rather high residual DHAPAT activity and the presence of processed thiolase, suggesting a milder clinical presentation (Gootjes et al., 2002). These findings correspond well with previous data that showed that the homozygous S600W mutation found in this cell line results in only a partial PTS1-protein import defect affecting the import of catalase and egfp-pts1 completely but the import of D-bifunctional protein and SCP2 only partly, while the import of acyl-coa oxidase was unaffected (Shimozawa et al., 1999; Stanley et al., 2006). Previous studies established that the PEX5 amino acid residues are involved in PTS1 recognition and that the PTS1-binding domain in PEX5 is formed by the two carboxy-terminal TPR motif clusters TPR1-3 and TPR5-7 and the 7C-loop (Figure 2). The two TPR motif clusters are thought to surround the PTS1 protein almost completely, while TPR4 forms a distinct hinge structure (Gatto et al., 2000). The 7C-loop is required to connect the two TPR clusters. In all cell lines we observed a defect in PTS1 protein import, which in most cases can be explained by the specific location of the mutations in the PTS1-binding domain (Figure 2). For example, the four missense mutations cause amino acid changes in either one of the TPR motifs (cell lines PEX5.1, 2, 3 and 6) or after the TPR motifs (cell line PEX5.4). Furthermore, cell line PEX5.5 has an in-frame deletion of exon 12, which in theory leads to the loss of TPR3 and TPR4. The remaining mutations result in truncated PEX5 proteins lacking the entire or part of the PTS1-binding domain (Table 3 and Figure 2). Four cell lines showed a defect in PTS1-protein import, whereas PTS2-protein import still occurred (PEX5.1-PEX5.4; Table 3). These cell lines all had missense mutations affecting amino acids that are located outside the domains previously shown to be involved in PTS2 protein import, including the amino acid residues required for PEX7 binding (Dodt et al., 2001; Matsumura et al., 2000), the pentapeptide repeats 2-4 required for binding PEX13, and the amino-terminal WxxxF/Y motifs required for binding PEX14 (Otera et al., 2002; Saidowsky et al., 2001) (Figure 2). Cell line PEX5.6 also has a missense mutation, R557W, that is not located in any of the domains that are known to be involved in PTS2 protein import, but this cell line 3 61

64 Chapter 3 3 does not show PTS2 protein import when transfected with GFP-PTS2. The reason for this is unclear. In the remaining cell lines, both PTS1- and PTS2-protein import was defective, indicating that the truncated PEX5 proteins in these cell lines are not functional, if stably expressed at all. Because the PEX5 protein expression levels in fibroblasts are too low to allow detection with commercially available antibodies raised against PEX5, this aspect could not be studied further. Remarkably, most of the detected mutations are located in the carboxy-terminal half of PEX5. This suggests that mutations in the amino-terminal half could give rise to a milder presentation, or to clinical and biochemical phenotypes that differ from those observed for Zellweger Syndrome. For example, a mutation in the PEX7-binding site could result in an RCDP-like phenotype typically observed in patients with defects in PEX7, whereas mutations in one of the multiple PEX14-binding sites may give rise to a partial protein import defect. For this reason, mutations in the PEX5 gene remain a possible cause for disease in patients with mild peroxisomal defects, similarly as we recently demonstrated for mild mutations in the PEX12 gene (MIM ; (Zeharia et al., 2007). Acknowledgements This study was supported by a grant from the Princes Beatrix Fonds and by the FP6 European Union Project peroxisomes (LSHG-CT ). References Braverman N, Dodt G, Gould SJ, Valle D An isoform of pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum Mol Genet 7: Brul S, Westerveld A, Strijland A, Wanders RJ, Schram AW, Heymans HS, Schutgens RB, van den Bosch H, Tager JM Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis. J Clin Invest 81: Carvalho AF, Grou CP, Pinto MP, Alencastre IS, Costa-Rodrigues J, Fransen M, Sa-Miranda C, Azevedo JE Functional characterization of two missense mutations in Pex5p - C11S and N526K. Biochim Biophys Acta 1773: Dodt G, Braverman N, Wong C, Moser A, Moser HW, Watkins P, Valle D, Gould SJ Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nat Genet 9: Dodt G, Warren D, Becker E, Rehling P, Gould SJ Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p. J Biol Chem 276: Gatto GJJr, Geisbrecht BV, Gould SJ, Berg JM Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct Biol 7: Gootjes J, Mooijer PA, Dekker C, Barth PG, Poll-The BT, Waterham HR, Wanders RJ Biochemical markers predicting survival in peroxisome biogenesis disorders. Neurology 59: Lazarow PB, Fujiki Y Biogenesis of peroxisomes. Annu Rev Cell Biol 1: Matsumura T, Otera H, Fujiki Y Disruption of the interaction of the longer isoform of Pex5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals. Study with a novel Pex5-impaired Chinese hamster ovary cell mutant. J Biol Chem 275: Motley AM, Brites P, Gerez L, Hogenhout E, Haasjes J, Benne R, Tabak HF, Wanders RJ, Waterham HR Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1. Am J Hum Genet 70: Ofman R, Wanders RJ Purification of peroxisomal acyl-coa: dihydroxyacetonephosphate 62

65 Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines acyltransferase from human placenta. Biochim Biophys Acta 1206: Otera H, Setoguchi K, Hamasaki M, Kumashiro T, Shimizu N, Fujiki Y Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import. Mol Cell Biol 22: Platta HW, Erdmann R The peroxisomal protein import machinery. FEBS Lett 581: Platta HW, El Magraoui F, Schlee D, Grunau S, Girzalsky W, Erdmann R Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling. J Cell Biol 177: Saidowsky J, Dodt G, Kirchberg K, Wegner A, Nastainczyk W, Kunau WH, Schliebs W The diaromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14. J Biol Chem 276: Shimozawa N, Zhang Z, Suzuki Y, Imamura A, Tsukamoto T, Osumi T, Fujiki Y, Orii T, Barth PG, Wanders RJ, Kondo N Functional heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5- defective patients. Biochem Biophys Res Commun 262: Stanley WA, Filipp FV, Kursula P, Schuller N, Erdmann R, Schliebs W, Sattler M, Wilmanns M Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p. Mol Cell 24: Stanley WA, Pursiainen NV, Garman EF, Juffer AH, Wilmanns M, Kursula P A previously unobserved conformation for the human Pex5p receptor suggests roles for intrinsic flexibility and rigid domain motions in ligand binding. BMC Struct Biol 7:24. Steinberg SJ, Dodt G, Raymond GV, Braverman NE, Moser AB, Moser HW Peroxisome biogenesis disorders. Biochim Biophys Acta 1763: van Grunsven EG, van Berkel E, Mooijer PA, Watkins PA, Moser HW, Suzuki Y, Jiang LL, Hashimoto T, Hoefler G, Adamski J, Wanders RJ Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis. Am J Hum Genet 64: Vreken P, van Lint AE, Bootsma AH, Overmars H, Wanders RJ, van Gennip AH Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry. J Chromatogr B Biomed Sci Appl 713: Wanders RJ, van Roermund CW Studies on phytanic acid alpha-oxidation in rat liver and cultured human skin fibroblasts. Biochim Biophys Acta 1167: Wanders RJ, Dekker C, Ofman R, Schutgens RB, Mooijer P. 1995a. Immunoblot analysis of peroxisomal proteins in liver and fibroblasts from patients. J Inherit Metab Dis 18 Suppl 1: Wanders RJ, Denis S, Ruiter JP, Schutgens RB, van Roermund CW, Jacobs BS. 1995b. Measurement of peroxisomal fatty acid beta-oxidation in cultured human skin fibroblasts. J Inherit Metab Dis 18 Suppl 1: Wanders RJ, Waterham HR Biochemistry of mammalian peroxisomes revisited. Annu Rev Biochem 75: Weller S, Gould SJ, Valle D Peroxisome biogenesis disorders. Annu Rev Genomics Hum Genet 4: Zeharia A, Ebberink MS, Wanders RJ, Waterham HR, Gutman A, Nissenkorn A, Korman SH A novel PEX12 mutation identified as the cause of a peroxisomal biogenesis disorder with mild clinical phenotype, mild biochemical abnormalities in fibroblasts and a mosaic catalase immunofluorescence pattern, even at 40 degrees C. J Hum Genet 52:

66 Chapter

67 Chapter 4 Spectrum of PEX6 Mutations in Zellweger Syndrome Spectrum Patients Merel S. Ebberink*, Janet Koster*, Ronald J.A. Wanders, and Hans R. Waterham University of Amsterdam, Academic Medical Center, Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Amsterdam, the Netherlands * These authors contributed equally to this study Human Mutation (2010) 31(1):E

68 Chapter 4 Abstract 4 The autosomal recessive Zellweger syndrome spectrum (ZSS) disorders comprise a main subgroup of the peroxisome biogenesis disorders. The ZSS disorders can be caused by mutations in any of 12 different currently identified PEX genes resulting in severe, often lethal, multi-systemic disorders. Defects in the PEX6 gene are the second most common cause for ZSS disorders. The encoded protein PEX6 belongs to the AAA ATPase family and contains two AAA cassettes and an AAA protein family signature. The PEX6 gene consists of 17 exons and previously mutations in the PEX6 gene were found to be scattered over all exons. We developed a post-pcr high-resolution melting (HRM) curve assay to scan the PEX6 gene for potential sequence variations followed by selective sequencing to identify these. We analyzed the PEX6 genes of 75 patients assigned to the PEX6 complementation group. We identified a total of 77 different mutations of which 47 mutations have not been reported previously, and 14 polymorphic variants. 66

69 Spectrum of PEX6 Mutations in ZSS Patients Introduction Human peroxisomes play an essential role in a variety of metabolic pathways, among which are the biosynthesis of ether phospholipids and the alpha- and betaoxidation of fatty acids (Wanders and Waterham, 2006). Peroxisome biogenesis disorders (PBDs; MIM ) are caused by a defect in genes encoding proteins required for assembly of the organelle, including proteins involved in the import of peroxisomal matrix and membrane proteins. The PBD group comprises two distinct clinical spectra: the Zellweger syndrome spectrum (ZSS) and the Rhizomelic Chondrodysplasia Punctata spectrum (RCDP; MIM ). The ZSS includes three defined phenotypes, namely the Cerebro-Hepato-Renal Syndrome of Zellweger (i.e. Zellweger Syndrome; ZS; MIM ), neonatal adrenoleukodystrophy (NALD; MIM ) and infantile Refsum disease (IRD; MIM ) with decreasing clinical and biochemical severity. Common to all three ZSS disorders are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness (Gould 2001, Weller 2008). In addition, patients with ZS are severely hypotonic from birth and die within their first year of life. Patients with NALD experience neonatal onset of hypotonia and seizures, have progressive white matter disease and usually die in late infancy. The survival of IRD patients is variable, with many patients surviving beyond infancy and some may even reach adulthood (Barth et al., 2001). IRD patients have no neuronal migration defect, but can develop a progressive white matter defect. The different phenotypes can be caused by mutations in different genes, as well as different mutations within the same gene, and show considerable clinical and biochemical overlap (Steinberg et al., 2006). 4 In humans, currently 13 different proteins, called peroxins, encoded by PEX genes have been shown to be essential for the biogenesis of peroxisomes (Steinberg et al., 2006). These peroxins are involved in various stages of import of proteins into peroxisomes and/or the assembly of these organelles. The autosomal recessive ZSS disorders can be caused by mutations in 12 of these PEX genes. To identify the underlying PEX gene defect in ZSS patients, different approaches have been described. These include a complementation assay based on fusion of a ZSS cell line by means of polyethylene glycol (PEG) treatment with a series of tester cell lines each representing a known PEX complementation group (Brul et al., 1988), complementation assays based on the transfection of ZSS cell lines with the known human PEX genes (Ebberink et al., 2009, see also chapter 2; Krause et al., 2009), and a systematic, hierarchical sequencing approach to detect mutations based on the frequency of PEX gene defects and mutations (Steinberg et al., 2004). A recent survey we conducted with more than 600 ZSS cell lines confirmed that defects in the PEX6 (MIM ) gene are the second most common cause for ZSS disorders (approximately 16%). PEX6 encodes a member of the AAA (ATPase Associated with diverse cellular Activities) ATPase family and is required for import of peroxisomal matrix proteins and/or vesicle fusion. Because the PEX6 gene consists of 17 exons and previously identified mutations in the PEX6 gene were found to be scattered over all exons, we set up a high-resolution melting curve (HRM) assay for fast mutation scanning of the PEX6 gene. This post-pcr HRM assay scans entire 67

70 Chapter 4 amplicons and detects potential sequence variations in these amplicons, after which the amplicons are sequenced to identify these. In this study, we analyzed the PEX6 genes of 75 patients previously assigned to the PEX6 complementation group. Materials and Methods 4 Preparation of gdna and cdna In primary skin fibroblasts from 75 patients we established a defect in the PEX6 gene using a PEX cdna transfection complementation assay to be reported elsewhere (Ebberink et al., 2009 and chapter 2). Genomic DNA was isolated from these skin fibroblasts using the Nucleospin Tissue kit (Macherey-Nagel, Düren, Germany). For some of the mutations, cdna analysis was performed to determine the effect of the mutations at the post-transcriptional level. To this end, total RNA was isolated from primary skin fibroblasts using Trizol (Invitrogen, Carlsbad, CA, USA) extraction, after which cdna was prepared using a first strand cdna synthesis kit for RT-PCR (Roche, Mannheim, Germany). Mutation analysis 16 Primer sets were designed to cover the complete coding region plus flanking intronic sequences of the PEX6 gene (primer sequences see Supp. Table S1). All forward and reverse primers were tagged with -21M13 (5 -TGTAAAACGACGGCCAGT-3 ) and M13rev (5 -CAGGAAACAGCTATGACC-3 ) extensions respectively. PCR amplifications were optimized in a volume of 10 µl containing 1µl 1x LC Green Plus (BioChem, Salt Lake City, Utah, USA) in 96-wells plates and analyzed on agarose gels to verify the correct size of the amplicons. The primers were used at a concentration of 0.25 μm. PCR reactions were carried out in duplicate. After amplification, the samples were heated to 95 C for 30 seconds and then cooled to 25 C for 30 seconds to allow heteroduplex formation. Exons 2 till 17 of the PEX6 gene from control subjects and the patient cells were analysed by post-pcr HRM analysis using the 96-well LightscannerTM (Idaho Technology, Salt Lake City, Utah, USA) to detect sequence variations. HRM curve data were obtained by melting over the desired range (70-98 C) at a rate of 25 acquisitions per 1 C. The amplicons that showed putative sequence variations Table 1. Post- PCR HRM scan efficiency. Exon HRM scan variation Mutations False positive Number of patient samples with a potential sequence variation detected with the HRM scan. 2 Number of patient samples that have mutations (including polymorphic variants) and showed sequence variation with the HRM scan. 3 Number of patient samples that have no mutations but showed sequence variation with the HRM scan. 68

71 Spectrum of PEX6 Mutations in ZSS Patients were sequenced using `-21M13` and `M13rev` primers by means of Big Dye Terminator v1.1 Cycle Sequencing (Applied Biosystems, Foster City, CA, USA) and analyzed on an Applied Biosystems 377A automated DNA sequencer, following the manufacturer s protocol (Applied Biosystems, Foster City, CA, USA). Exon 1 was directly sequenced after amplicon amplification, because this exon is known to contain multiple polymorphisms making post PCR HRM analysis problematic. The PEX6 sequences were compared to the reference sequence of PEX6 (GenBank accession number NM_ ) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. Results and Discussion We set up a rapid mutation scanning method for the PEX6 gene. This post-pcr HRM assay scans 14 amplicons and detects potential sequence variations with different efficiencies. Using this assay, we found on average 3 amplicons per patient that showed potential sequence variations (with a maximum of 7 amplicons per patient). Subsequent sequence analysis of these aberrant amplicons to confirm the presence of mutations showed that for 5 amplicons the scan was very reliable (no false positives), whereas in other amplicons mutations were not always detected (Table 1). In 3 patients cell lines we could not identify a mutation in the amplicons that showed potential sequence variations; in these cell lines we sequenced the remainder of the gene. In our study, we analyzed the PEX6 gene of 75 different PEX6-defective cell lines. In 27 patient cell lines mutation analysis was already performed before we developed the post-pcr HRM-assay. Sequencing exon 1 in the remaining 48 patient cell lines identified 7 homozygous mutations and 10 heterozygous mutations (of which 2 in one patient cell line). Post-HRM analysis was subsequently performed with the remaining 40 cell lines. By using the post-pcr HRM scan, we obtained a significant reduction in the total number of sequence reactions required to analyze the PEX6 gene. Instead of the 640 sequence reactions that would have been required to analyze the entire PEX6 gene in the 40 patients subjected to post-pcr HRM analysis, we now only had to perform 240 sequence reactions, including 80 sequence reactions required for analysis of exon 1. We identified a total of 77 different mutations of which 47 were heterozygous and 30 homozygous. Of the 77 mutations, 47 mutations have not been reported previously. Combined with previously reported PEX6 mutations, the PEX6 mutational spectrum now comprises 88 different mutations (Table 2 and Supp. Table S2). In addition, 14 polymorphic variants were identified in the PEX6 gene (Table 3). Of the 88 different mutations, 35 are missense (40%), 4 are nonsense (5%), 24 are deletions (27%), 2 are insertions (3%), 1 is a insertion-deletion (1%), 4 are duplications (4%) and 18 are substitutions in splice junction sequences (20%). Among the 47 novel mutations identified in our cohort, there were 11 mutations located in splice junction sequences. The c g>a mutation was found homozygous in 2 different cell lines. PEX6 cdna analysis revealed that this mutation causes skipping of exon 3 (p.delv350_r377) and the insertion of the last 20 base pairs of intron 2, which results in a frame shift (p.v350fsx7). The c a>g 4 69

72 Chapter 4 Table 2. Mutation spectrum of the PEX6 gene. 4 Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygouzygous Hetero- Reference 1 c.170t>c p.l57p 1 1 (a) 1 c.224dubt p.v76gfsx2 1 This paper 1 c.275_280deltgcggg p.v92_r93del 1 (b) 1 c.277c>g p.r93g 1 This paper 1 c.281c>a p.a94k (c) 1 c.311delg p.g104vfsx22 1 This paper 1 1 c.35t>c p.f12s (c) 1 c.402delc p.g135fsx22 6 (a, b) 1 c.510_511inst p.g171fsx70 (d) 1 c.517dela p.s173fsx32 1 (c) 1 c.530delc p.p177fsx28 1 (b) 1 c.557c>a p.a186e 1 This paper 1 c.656a>c p.q219p 1 This paper 1 c.656dela p.q219rfsx27 1 This paper 1 c.659g>t p.g220v 1 This paper 1 c.689_670delag p.e230vfsx11 1 This paper 1 c.690_691dupag p.s232hfsx15 1 (e) 1 c.727c>t p.q243x (b) 1 c.802_815del p.v207_q294del 1 (f) 1 c.814_817dupcttg p.v273fsx8 1 (b) 1 c.821c>t p.p274l 1 2 (c) 1 c.856delc p.l286wfsx65 1 This paper 1 c.867dela p.e290sfsx61 1 This paper 1 c.882+1g>a (c) 2 c.883-2a>g p.r295fsx34/x8 (b) 2 c.914dela p.d305fs This paper 2, 3 c.883_1130del p.r295vfsx7 2 This paper 3 c g>a p.v350fsx7, p.v350_ 2 This paper R377 3 c A>G p.v350_r377 1 This paper 3 c.1054c>t p.v350_r377del 1 This paper 3 c g>a p.v350_w378del (d) 4 c g>c p.r379fsx3 1 This paper 4 c.1198t>a p.y400n 1 This paper 5 c.1301delc p.s343ffsx16 (b) 5 c.1314_1321delggaggcct p.e439gfsx3 5 8 (e) 5 c delg 1 This paper 6 c.1404dela p.r469gfsx11 1 This paper 6 c.1415dupc p.g473rfsx13 (e) 6, 7 c g>a [p.v494fsx18, p.g457_ 1 (b) S563del] 7 c.1495delc p.l499sfsx49 1 This paper 7 c.1532t>g p.l511r 1 This paper 7 c.1553c>a p.a518d 1 This paper 7 c g>a [p.v494fsx18, p.g457_ 1 (b) S563del] 8 c g>t p.s563rfsx5 1 This paper 8 c.1711g>a p.a571t 1 This paper 8 c.1715c>t p.t572i 1 1 (h) 8 c.1793_1794delag p.e598gfsx63 1 This paper 8 c.1801c>t p.r601w 1 1 This paper 70

73 Spectrum of PEX6 Mutations in ZSS Patients Table 2. Continue. Mutations Number of patients identified in this study Exon Nucleotide Amino acid Homozygouzygous Hetero- Reference 8 c.1802g>a p.r601q 4 (g) 8 c.1814t>g p.l605r 1 This paper 9 c.1947delg p.i650fsx (e) 9 c.1958c>g p.s653x 1 This paper 9 c g>a p.l655fsx3 (i) 10 c.1992g>c p.e664d 1 This paper 10 c.2048t>c p.l683p (e) 10 c t>c p.i699fsx37 1 (h) 10 c _21del [p.ile699ins7fs39x, (c) p.ile699fs39x] 10 c.2120t>g p.v707g (e) 11 c a>g Incorrect splicing 1 This paper 11 c p.r736tfsx21 1 This paper 2217delGACGCTCAGGC 11 c.2225t>c p.l742p 1 This paper 12 c c>g 2 This paper 11 c c>g 4 splice products 1 This paper 12 c g>a p.k769fsx8 1 This paper 12 c.2345g>a p.r812q (b) 12 c.2362g>a p.v788m of I699fs739X 1 (f) 12 c.2356c>t p.r786w 1 This paper 12 c.2362g>a p.i699fsx (f) 13 [c.2426c>t, c.2534t>c] p.a809v, p.i845t (h) 13 c.2398_2417delinst p.i800fsx15 1 (i) 13 c.2434c>t p.r812w 3 (b) 13 c.2435g>a p.r812q 1 2 (b) 13 c.2440c>t p.r814x 1 2 This paper 14 c.2482c>t p.q828x 1 This paper 14 c.2546a>c p.n849t (g) 14 c.2578c>t p.r860w (g) 14 c.2579g>a p.r860q (g) 15 c.2591t>c p.f864s 1 This paper 15 c.2602ins p.v868fsx31 2 This paper 15 c.2626c>t p.r876w 1 This paper 15 c.2663g>c p.r888p 1 This paper 15 c t>c p.d865_f890del (b) no PCR-product exon This paper 16 c a>g 1 This paper 16 c.2726t>a p.l909q 2 This paper 17 c a>g p.l937fsx8 1 This paper 1 Mutation is mentioned in the dbpex database ( The PEX6 sequences were compared to the reference sequence of PEX6 (GenBank accession number NM_ ) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. 4 (a) Imamura et al., 2000 (d) Fukuda et al., 1996 (g) Yik et al., 2009 (b) Zhang et al., 1999 (e) Krause et al., 2006 (h) Raas-Rothschild et al., 2002 (c) Steinberg et al., 2004 (f) Matsumoto et al., 2001 (i) Yahraus et al.,

74 Chapter 4 4 mutation results in skipping of exon 3 although a small proportion of correctly sized PEX6 cdna was observed. The c g>c and c g>t mutations result in skipping of exon 4 and exon 8, respectively. The c c>g mutation gives rise to four different mrna products. The c g>a and c a>g mutations cause the insertion of a part of intron 11 and intron 16, respectively, which results in a frame shift (p.k769fsx8 and p.l937fsx8). We could not analyze the effect of the c delg, c c>g and c a>g mutations, because there were no viable cells from these patients available, but we expect for all three mutations that they result in incorrect splicing. PEX6 belongs to the AAA ATPase family, which comprises proteins that share a region of amino acids, containing two AAA cassettes and an AAA protein family signature. Each AAA cassette includes Walker A (amino acids and ) and Walker B (amino acids and ) nucleotide binding motifs. The second Walker A motif is essential for biological activity of PEX6 (Yahraus et al., 1996). Ten of the eighteen novel missense mutations are located in the AAA cassette domains, of which three mutations (p.l511r, p.a518d and p.r786w) are located in one of the two Walker B motifs and one mutation (p.f864s) is located in the AAA protein family signature. All missense mutations are not listed in the SNP database, have not been detected in more than 200 control chromosomes and in fact all affect well conserved amino acids. Alignment studies were performed by using the computer software Alamut (version 1.5 rev. 32) of Interactive Biosoftware. This program performs a protein multi-alignment of the PEX6 proteins of Homo sapiens (human), Pan troglodytes (chimpanzee), Rattus rattus (rat), Mus musculus (mouse), Canis familiaris (dog), Gallus gallus domesticus (chicken), Xenopus tropicalis (frog), Tetraodon nigroviridis (pufferfish), Drosophila melanogaster (fruitfly), Caenorhabditis elegans (roundworm) Table 3. Indentified SNPs and neutral variants in the PEX6 gene. Exon Intron Nucleotide Amino acid SNP Frequency 1 Reference database c.1-55c>t No 0.54 this paper 1 c.207c>t p.p69p Yes this paper 1 c.210g>a p.g70g No 0.01 (a) 1 c.235g>c p.a79p Yes (b) 1 c.330c>g p.t110t No 0.02 this paper 1 c.399g>t p.v133v Yes 0.53 (a,b) 1 c.423a>g p.t141t No 1.0 this paper 1 c.883-3t>c Yes 0.08 this paper 7 c c>t Yes this paper 9 c g>a No this paper 13 c.2364g>a p.v768v Yes 0.03 (a,b) 13 c.2425c>t p.a809v Yes (a,b) 17 c.2814g>a p.e938e Yes 0.20 (a) 17 c.2816c>a p.p939q Yes 0.17 (a) 1 Frequency based on the analysis of 126 alleles. (a) Yik et al., 2009 (b) Steinberg et al.,

75 Spectrum of PEX6 Mutations in ZSS Patients and Saccharomyces cerevisiae (baker s yeast). The p.r93g, p.q219p, p.g220v, p.e664d, p.r876w and p.l909q mutations affect amino acids conserved in mouse, rat, chimpanzee, dog, mouse, and rat. The p.a186e, p.l511r, p.a518d, p.a571t, p.r601w, p.l605r, pl742p, p.r786w, p.f864s and R888P mutations are amino acids conserved in PEX6 of mouse, rat, chimpanzee, dog, Arabidopsis thaliana (thale cress) and/or baker s yeast. The p.y400n mutation affects an amino acid conserved in PEX6 of chimpanzee, dog, chicken, frog and pufferfish. Interestingly, although the heterozygous c.1054c>t mutation theoretically results in p.q352x, it appeared that the consequence at the mrna level is skipping of exon 3. Nine of the ten novel deletion mutations identified in our cohort result in a frame shift (Table 2). One in-frame deletion (p.a39_l40del) was detected. From one patient cell line we could not amplify exons 14, 15 and 16 which indicates a large intragenic deletion. The three novel nonsense mutations (p.s653x, p.r814x and p.r814x) result in a truncated PEX6 protein lacking an intact AAA cassette domain. Of the fourteen polymorphic variants we identified in the PEX6 gene (Table 3), 4 were located in non-coding regions (c.1-55c>t, c.883-3t>c, c c>t and c g>a), 7 were silent (c.207c>t, c.210g>a, c.330c>g, c.399g>t, c.423a>g, c.2364g>a and c.2814g>a), and three resulted in an amino acid substitution. (c.235g>c, c.2425c>t and c.2816c>a). The variants are assumed to be without pathogenic consequence on the basis of their occurrence in multiple individuals, including healthy controls. In addition, nine of the 14 variants are listed in the SNP database as commonly occurring polymorphic variants. 4 Acknowledgement This study was supported by a grant from the Prinses Beatrix Fonds (MAR 03_0216) and the FP6 European Union Project peroxisomes (LSHG-CT ). We thank Dr. Alders for assistance in setting up the post-pcr HRM analysis. Supplemental Data References Braverman N, Dodt G, Gould SJ, Valle D An isoform of pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum Mol Genet 7: Brul S, Westerveld A, Strijland A, Wanders RJ, Schram AW, Heymans HS, Schutgens RB, van den BH, Tager JM Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis. J Clin Invest 81: Carvalho AF, Grou CP, Pinto MP, Alencastre IS, Costa-Rodrigues J, Fransen M, Sa-Miranda C, Azevedo JE Functional characterization of two missense mutations in Pex5p - C11S and N526K. Biochim Biophys Acta 1773: Dodt G, Braverman N, Wong C, Moser A, Moser HW, Watkins P, Valle D, Gould SJ Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nat Genet 9:

76 Chapter 4 4 Dodt G, Warren D, Becker E, Rehling P, Gould SJ Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p. J Biol Chem 276: Gatto GJJr, Geisbrecht BV, Gould SJ, Berg JM Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct Biol 7: Gootjes J, Mooijer PA, Dekker C, Barth PG, Poll-The BT, Waterham HR, Wanders RJ Biochemical markers predicting survival in peroxisome biogenesis disorders. Neurology 59: Lazarow PB, Fujiki Y Biogenesis of peroxisomes. Annu Rev Cell Biol 1: Matsumura T, Otera H, Fujiki Y Disruption of the interaction of the longer isoform of Pex5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals. Study with a novel Pex5-impaired Chinese hamster ovary cell mutant. J Biol Chem 275: Motley AM, Brites P, Gerez L, Hogenhout E, Haasjes J, Benne R, Tabak HF, Wanders RJ, Waterham HR Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1. Am J Hum Genet 70: Ofman R, Wanders RJ Purification of peroxisomal acyl-coa: dihydroxyacetonephosphate acyltransferase from human placenta. Biochim Biophys Acta 1206: Otera H, Setoguchi K, Hamasaki M, Kumashiro T, Shimizu N, Fujiki Y Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import. Mol Cell Biol 22: Platta HW, Erdmann R The peroxisomal protein import machinery. FEBS Lett 581: Platta HW, El MF, Schlee D, Grunau S, Girzalsky W, Erdmann R Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling. J Cell Biol 177: Saidowsky J, Dodt G, Kirchberg K, Wegner A, Nastainczyk W, Kunau WH, Schliebs W The diaromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14. J Biol Chem 276: Shimozawa N, Zhang Z, Suzuki Y, Imamura A, Tsukamoto T, Osumi T, Fujiki Y, Orii T, Barth PG, Wanders RJ, Kondo N Functional heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5- defective patients. Biochem Biophys Res Commun 262: Stanley WA, Filipp FV, Kursula P, Schuller N, Erdmann R, Schliebs W, Sattler M, Wilmanns M Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p. Mol Cell 24: Stanley WA, Pursiainen NV, Garman EF, Juffer AH, Wilmanns M, Kursula P A previously unobserved conformation for the human Pex5p receptor suggests roles for intrinsic flexibility and rigid domain motions in ligand binding. BMC Struct Biol 7:24. Steinberg SJ, Dodt G, Raymond GV, Braverman NE, Moser AB, Moser HW Peroxisome biogenesis disorders. Biochim Biophys Acta 1763: van Grunsven EG, van BE, Mooijer PA, Watkins PA, Moser HW, Suzuki Y, Jiang LL, Hashimoto T, Hoefler G, Adamski J, Wanders RJ Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis. Am J Hum Genet 64: Vreken P, van Lint AE, Bootsma AH, Overmars H, Wanders RJ, van Gennip AH Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry. J Chromatogr B Biomed Sci Appl 713: Wanders RJ, van Roermund CW Studies on phytanic acid alpha-oxidation in rat liver and cultured human skin fibroblasts. Biochim Biophys Acta 1167: Wanders RJ, Dekker C, Ofman R, Schutgens RB, Mooijer P. 1995a. Immunoblot analysis of peroxisomal proteins in liver and fibroblasts from patients. J Inherit Metab Dis 18 Suppl 1: Wanders RJ, Denis S, Ruiter JP, Schutgens RB, van Roermund CW, Jacobs BS. 1995b. Measurement of peroxisomal fatty acid beta-oxidation in cultured human skin fibroblasts. J Inherit Metab Dis 18 Suppl 1: Wanders RJ, Waterham HR Biochemistry of mammalian peroxisomes revisited. Annu Rev Biochem 75: Weller S, Gould SJ, Valle D Peroxisome biogenesis disorders. Annu Rev Genomics Hum Genet 4: Zeharia A, Ebberink MS, Wanders RJ, Waterham HR, Gutman A, Nissenkorn A, Korman SH A novel PEX12 mutation identified as the cause of a peroxisomal biogenesis disorder with mild clinical phenotype, mild biochemical abnormalities in fibroblasts and a mosaic catalase immunofluorescence pattern, even at 40 degrees C. J Hum Genet 52:

77 Chapter 5 Identification of an unusual Variant Peroxisome Biogenesis Disorder Caused by Mutations in the PEX16 Gene Merel S. Ebberink 1, Barbara Csanyi 2, Wui K. Chong 3, Simone Denis 1, Peter Sharp 4, Petra A.W. Mooijer 1, Conny J.M. Dekker 1, Claire Spooner 5, Lock H. Ngu 6, Carlos De Sousa 7, Ronald J.A. Wanders 1, Michael J. Fietz 4, Peter T. Clayton 2, Hans R. Waterham 1, Sacha Ferdinandusse 1 1 Academic Medical Centre, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Departent of Paediatrics/Emma Children s Hospital, Amsterdam, The Netherlands; 2 Biochemistry Research Group, UCL Institute of Child Health, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom, 3 Department of Radiology, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom, 4 National Referral Laboratory, SA Pathology, North Adelaide, Australia, 5 Starship Children s Hospital, Auckland District Health Board, New Zealand, 6 Genetics Department, Kuala Lumpur Hospital, Kuala Lumpur, Malaysia, 7 Department of Neurology, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom Journal of Medical Genetics (2010)

78 Chapter 5 Abstract 5 Objective Zellweger syndrome spectrum disorders are caused by mutations in any of at least 12 different PEX genes. This includes PEX16, which encodes an integral peroxisomal membrane protein involved in peroxisomal membrane assembly. PEX16-defective patients have been reported to have a severe clinical presentation. Fibroblasts of these patients displayed a defect in import of peroxisomal matrix and membrane proteins, resulting in a total absence of peroxisomal remnants. Here, we report 6 patients with an unexpected mild variant peroxisome biogenesis disorder due to mutations in the PEX16 gene. Patients presented in the preschool years with progressive spastic paraparesis and ataxia (with a characteristic pattern of progressive leukodystrophy and brain atrophy on MRI scan) and later developed cataracts and peripheral neuropathy. Surprisingly, their fibroblasts showed enlarged, import-competent peroxisomes. Results Plasma analysis revealed biochemical abnormalities suggesting a peroxisomal disorder. Biochemical parameters in fibroblasts were only mildly abnormal or within the normal range. Immunofluorescence microscopy analyses revealed the presence of import-competent peroxisomes, which were increased in size but reduced in number. Subsequent sequencing of all known PEX genes revealed five novel apparent homozygous mutations in the PEX16 gene. Conclusion We identified an unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene, with a relatively mild clinical phenotype and an unexpected phenotype in fibroblasts. Although PEX16 is involved in peroxisomal membrane assembly, PEX16 defects can present with enlarged import-competent peroxisomes in fibroblasts. This is important for future diagnostics of patients with a peroxisomal disorder. 76

79 Identification of an unusual Variant PBD Introduction The Zellweger Syndrome Spectrum (ZSS), including Zellweger Syndrome (ZS, MIM ), neonatal adrenoleukodystrophy (NALD, MIM ) and infantile Refsum disease (IRD, MIM ), comprises a spectrum of severe, often lethal, inherited multisystemic disorders. Variable neurodevelopmental delay, liver disease, retinopathy and perceptive deafness are characteristic for the disorders within the ZSS. ZS is the most severe disorder within this spectrum. ZS patients have profound neurological abnormalities and display typical craniofacial dysmorphia (Gould et al., 2001). ZS patients generally die within the first year of life. Like ZS patients, patients with NALD suffer from neonatal hypotonia and seizures. They may suffer from progressive white matter disease, and usually die in late infancy (Wanders and Waterham, 2005). Patients with IRD have no neuronal migration defect, but can develop a progressive white matter defect. Their survival is variable, but most patients survive beyond infancy and some even reach adulthood (Poll-The BT et al., 1987). The disorders of the ZSS are characterized by the absence of functional peroxisomes and a generalized loss of peroxisomal functions. Defects in any of at least 12 different peroxins (PEX), encoded by PEX genes, have been found to result in a peroxisome biogenesis disorder (PBD, MIM ) of the ZSS type. Peroxins are involved in the import of proteins into the peroxisome and/or the biogenesis of these organelles. Different mutations in the same PEX gene can lead to different phenotypes within the spectrum. PEX1, PEX2, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14 and PEX26 are involved in the import of peroxisomal matrix proteins. A defect in one of the genes encoding these peroxins results in impaired peroxisomal matrix protein import, but peroxisomal membrane structures (peroxisomal ghosts) are still present. In contrast, fibroblasts with a defect in the genes encoding PEX3, PEX16 or PEX19, which are involved in the peroxisomal membrane protein import, were shown to have no peroxisomal remnants at all (Honsho et al., 1998; Shimozawa et al., 2000). Human PEX16 is an integral peroxisomal membrane protein (PMP) with two membrane-spanning domains. So far, only three patients have been reported with a defect in PEX16, all displaying the severe ZS phenotype. Fibroblasts of these patients showed a complete lack of peroxisomes, including membranes, and peroxisomal functions (Honsho et al., 1998; Shimozawa et al., 2002). In this paper, we report 6 patients, including one sib pair, who all have a defect in PEX16, but who showed enlarged, protein import-competent peroxisomes in their fibroblasts. 5 Patients and Methods Patients Patient 1, a girl, was the first child of consanguineous Turkish parents. She was born at term with a normal birth weight and Apgar scores. She had no dysmorphic features and developed normally until 9 months of age. She started with toe walking at 13 months. Her walking remained unsteady with frequent falls. On examination at the age of 3 years, she presented a normal mental status, a bilateral horizontal 77

80 Chapter 5 5 nystagmus, increased tone in lower limbs, but normal in upper limbs, normal sensation, ataxia, slight dysmetria, a head and bilateral hand tremor, very brisk tendon reflexes at the knees, extensor plantar reflex and sustained bilateral ankle clonus. She showed no cognitive impairment, no organomegaly and no cranial nerve abnormalities. She stopped walking completely at the age of 5 years and ever since has been wheelchair bound. At the age of 10 years she developed dysarthria and dysphagia, and from that time she required full gastrostomy feeding. At the age of 13 years, ophthalmological examination revealed optic atrophy and very mild lens opacities. At the age of 14 years she was admitted to a hospital due to constipation and severe neuropathic pain. On examination she had normal sensory responses to touch, temperature and pain, but decreased vibration sense (absent in the ankles). Neurophysiology studies revealed evidence of a progressive demyelinating motor and sensory neuropathy affecting upper and lower limbs without axonal involvement on electroneuromyography (ENMG). By the age of 16 years she was suffering from spasticity in all four limbs and had no independent mobility. Her cataracts had become sufficiently dense as to require surgical removal. Repeated electroretinograms (ERGs) revealed normal flash responses suggesting normal function of the outer retinal receptor layer, whereas the visual evoked potentials (VEPs) showed small and very delayed potentials suggesting marked impairment of the visual pathways bilaterally. There have never been any concerns about her hearing. Magnetic resonance imaging (MRI) scans were performed at the age of 4, 6 (Figure 1A), 15 and 17 years. They showed extensive, diffuse and symmetrical signal abnormalities of myelinated white matter in the form of increased signal on T2-weighted images and near normal signal on T1-weighted images. These changes initially involved the dorsal brainstem, internal capsules (more severely in the posterior limb) and deep peritrigonal and parietal white matter, and spared the corpus callosum and subcortical U-fibres of white matter. There was no evidence of a malformation of cortical development. Later scans showed extension of disease into the corpus callosum and the subcortical white matter with some reduction in signal of the involved regions of white matter on T1-weighted images, whilst remaining more severely abnormal on T2-weighted images. There was generalized prominence of the ventricles and sulci in line with reduction in cerebral volume, however at the same time, more selective atrophy of the corpus callosum and cerebellar vermis was also observed. Patient 2 is the six years younger brother of patient 1, who was born at term with normal birth weight. He started to walk independently at the age of 17 months, despite lower limb spasticity, with an unsteady gait and frequent falls. He ceased independent walking at 25 months of age and was wheelchair bound from the age of 3 years. On examination at the age of 4 years, he had rigidity particularly in his lower limbs, brisk reflexes, progressive upper limb tremor, dysmetria and dysarthria. A neuropathy became apparent from the age of 5 years with increasing difficulty in emptying his bladder and worsening constipation. At the age of 10 years a demyelinating motor and sensory neuropathy was shown with ENMG and he developed dysaesthesia in his legs. As for his sister, his flash ERG responses were normal, whereas the VEP responses were delayed and not very well formed. Cataract was demonstrated. 78

81 Identification of an unusual Variant PBD 5 Figure 1. MRI brain images Axial T2-weighted, coronal and sagittal T1-weighted MRI brain images of patient 1 at the age of 6 years (A). Axial T2-weighted, coronal and sagittal T1-weighted MRI brain images of patient 2 at the age of 3 years (B). Axial T2-weighted and sagittal T1-weighted MRI brain images of patient 2 at the age of 11 years (C). Axial T2-weighted and sagittal T1-weighted MRI brain images of a normal child, annotated to show the dorsal brainstem (white arrow), posterior limb of the internal capsule (black arrow), corpus callosum (black arrow head) and cerebellar vermis (white arrow head) (D). All the patient studies showed a pattern of leukodystrophy where the abnormalities were more obvious on the T2-weighted images and less obvious on the T1-weighted images. Atrophy was global but also particularly notable in the corpus callosum and cerebellar vermis. Currently, he is 11 years old and his cognition is relatively spared. His MRI findings (Figure 1B and 1C) resembled those of his sister, showing a similar pattern of progressive leukodystophy and brain atrophy from the age of 3 years. The case report of patient 3 was published previously (Pineda et al., 1999). No detailed clinical information is available for patient 4. Patient 5, a girl, was born following an uneventful pregnancy and delivery. Her development was normal in the first year of life and she started walking independently at the age of months. At the age of two years she developed an ataxic gait. At the age of 6 years, she had mild cognitive impairment, moderate dysarthria and abnormal eye saccades, but no problems with speech or swallowing. MRI scans 79

82 Chapter 5 revealed widespread white matter changes on a background pattern of global delay in myelin maturation, with symmetrical white matter abnormality of the dorsal brainstem and capsular white matter, sparing of the corpus callosum and appearing more extensive and conspicuous on T2-weighted images than on T1-weighted images. There was reduced cerebellar volume, more severely affecting the vermis. Similar MRI findings were observed in the sister of patient 5 who was two years younger. 5 Patient 6, a girl, is the second child of consanguineous parents of Indian ethnicity. She was born at term with a normal birth weight. Her development was normal during the first year of life. She walked independently at 13 months and spoke a few short phrases at 18 months. After this she was noted to lose previously acquired skills and did not gain new skills. She lost her ability to walk independently at 24 months due to a combination of spasticity and mild ataxia. Her speech and other cognitive functions also deteriorated slowly over time. At the age of 5 years, nystagmus and cataracts in both eyes were observed. In her lower limbs, the muscular tone was increased, reflexes were brisk, clonus was present and plantar reflexes were upgoing. She had mild cerebellar signs. She did not have organomegaly. At 6 years, her cerebral white matter was noted to be diffusely hyperintense compared to grey matter on T2-weighted images in nearly all areas, particularly the deep and capsular white matter. The posterior fossa and callosal white matter was isointense with grey matter; that is, relatively spared. At the same time, all except the subcortical white matter was hyperintense compared to grey matter on T1-weighted images (that is, the comparatively normal appearance of myelinated white matter on these images). In addition, there was evidence of more focal atrophy of the cerebellum and corpus callosum. Peripheral nerve velocity studies of the lower limbs were suggestive of demyelination. Currently, at the age of 9, she is able to stand with a supportive frame, communicate in short sentences, and she is able to read and write simple sentences. Skin fibroblasts of the patients used in this study were sent to the Laboratory Genetic Metabolic Diseases at the Academic Medical Center of the University of Amsterdam for diagnostic purposes and informed consent was obtained for publication of the data. Cell culturing Primary skin fibroblasts were cultured in DMEM medium with 4.5 g/l glucose, L- glutamine (BioWhittaker, Lonza, Verviers, Belgium) and 25 mm Hepes, or in HAM F-10 medium with L-glutamine and Hepes 25 mm (Gibco, Invitrogen, Carlsbad, CA), each supplemented with 10% fetal bovine serum (FBS, BioWhittaker), 100 U/ml penicillin, 100 µg/ml streptomycin, in a humidified atmosphere of 5% CO 2, at 37 C. DMEM medium was used for the transfection experiments and HAM F-10 medium for the biochemical experiments. Biochemical analysis Levels of very long chain fatty acids (VLCFAs), phytanic and pristanic acid, and C 27 - bile acid intermediates were measured in plasma as described before (Dacremont 80

83 Identification of an unusual Variant PBD et al., 1995). Plasmalogens were determined in erythrocytes as previously described (Dacremont and Vincent, 1995). Dihydroxyacetonephosphate acyltransferase (DHAPAT; Ofman and Wanders, 1994), acyl-coa oxidase I (AOXI, Wanders et al., 1993) and D-bifunctional protein (DBP; van Grunsven et al., 1999) activity, concentrations of VLCFAs (Vreken et al., 1998), β-oxidation of C26:0, C16:0 and pristanic acid (Wanders et al., 1995b) and α-oxidation of phytanic acid (Wanders and van Roermund, 1993) were measured in cultured fibroblasts as previously described. Catalase immunofluorescence and immunoblot analysis using antibodies against peroxisomal thiolase 1, AOXI, and DBP were performed as described (van Grunsven et al., 1999; Wanders et al., 1995a). Mutation analysis Mutation analysis was performed by either sequencing all exons plus flanking intronic sequences of the PEX gene amplified by PCR from genomic DNA or by sequencing cdnas prepared from total mrna fractions. Genomic DNA was isolated from skin fibroblasts using the NucleoSpin Tissue genomic DNA purification kit (Machereynagel, Germany, Düren). Total RNA was isolated from skin fibroblasts using Trizol (Invitrogen, Carlsbad, CA) extraction, after which cdna was prepared using a first strand cdna synthesis kit for RT-PCR (Roche, Mannheim, Germany). All forward and reverse primers (PEX16 primer sequences are listed in supplemental table 1) were tagged with a -21M13 (5- TGTAAAACGACGGCCAGT-3 ) sequence or M13rev (5 -CAGGAAACAGCTATGACC-3 ) sequence, respectively. PCR fragments were sequenced in two directions using -21M13 and M13rev primers by means of BigDye Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA) and analyzed on an Applied Biosystems 377A automated DNA sequencer, following the manufacturer s protocol (Applied Biosystems, Foster City, CA, USA). Mutation analysis of PEX1 and PEX3 was performed by sequencing of cdna. Mutations in the PEX2 gene were identified by sequencing exon 4 from gdna. Mutation analysis of PEX5L, PEX6, PEX10, PEX12, PEX13, PEX14, PEX19 and PEX26 were performed by sequencing of all exons plus flanking intronic sequences. Mutation analysis of the PEX16 gene was performed by sequencing the PEX16 cdna. Mutations were confirmed by sequencing the corresponding exons and flanking intronic sequences of the PEX16 gene. Sequences were compared to the reference PEX16 sequence (NM_004813) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. 5 Functional complementation assay Fibroblasts of the five unrelated patients (2-6) were co-transfected with a pcdna3 expression plasmid containing either the PEX16 cdna (transcription variant 1 containing exon 11a, NM_ ) or, as a control, the PEX12 cdna (gift of Dr. S.J. Gould), and the pdsred-express-dr vector (Clontech Laboratories; used to identify transfected cells) using the AMAXA nucleofector technology (Amaxa, Cologne, Germany). The fibroblasts were examined by catalase immunofluorescence 72 hours after transfection (van Grunsven et al., 1999). Two independent transfections per construct were examined by counting the number of peroxisomes in at least 100 cells. Similar sized cells with the nucleus in focus were counted under the microscope using a counter. 81

84 Chapter 5 5 Table 1. Biochemical parameters in plasma and erythrocytes. VLCFAs VLCFAs Branched-chain fatty acids Bile acid intermediates Plasmalogens C26/C22 C26:0 Phytanic acid Pristanic acid DHCA THCA DMA C16:0 DMA C18:0 (ratio) (μm) (μm) (μm) (μm) (μm) (% of total phospholipids) Control range Patient n.d Patient Patient ND ND ND Patient Patient <0.1 <0.1 ND ND Patient ND ND ND ND Plasmalogens were determined in erythrocytes, all other parameters in plasma. VLCFAs = very long chain fatty acids; DHCA = dihydroxycholestanoic acid; THCA = trihydroxycholestanoic acid; DMA = dimethylacetal; n.d. = not detectable; ND = not determined. Table 2. Biochemical parameters in cultured skin fibroblasts. VLCFAs VLCFAs β-oxidation α-oxidation Enzyme activity C26/C22 C26:0 C26:0 Pristanic acid Phytanic acid DHAPAT AOXI DBP (ratio) (μmol/g protein) (pmol/(hr*mg) protein) (pmol/(hr*mg) protein) (nmol/(2hr*mg) protein) (pmol/min/mg) Control range ±29 252±79 Patient Patient Patient Patient ND ND Patient ND ND VLCFAs = very long chain fatty acids; DHAPAT = dihydroxyacetonephosphate acyltransferase; AOXI = acyl-coa oxidase I; DBP = D-bifunctional protein; ND = not determined. 82

85 Identification of an unusual Variant PBD Expression of mutant PEX16 cdna The mutant PEX16 cdnas of patient 1-4 were amplified by PCR from cdna prepared from mrna isolated from the patient fibroblasts and subcloned in the mammalian expression vector pcdna3 (Invitrogen). The wild type and mutant PEX16 cdnas were separately expressed in a previously reported PEX16-deficient fibroblast cell line (homozygous for an R298fsX38 mutation) with complete lack of peroxisomal structures (Shimozawa et al., 2002) and a control fibroblast cell line. Four days after transfection the fibroblasts were examined by catalase immunofluorescence to assess the appearance of peroxisomes. Two independent transfections per construct were examined by counting the number of peroxisomes in at least 100 cells. Similar sized cells with the nucleus in focus were counted under the microscope using a counter. To control for transfection efficiency the constructs were also co-transfected with a GFP-SKL expression vector in parallel experiments. The transfection efficiency was comparable for the different constructs. Results 5 Biochemical analysis The major presenting symptom in all cases described for the first time in this paper was difficulty with walking at around 2 years due to a combination of spasticity and ataxia. Presentation with a leukodystrophy at this age has been seen previously in a few children with disorders of peroxisome biogenesis. For this reason, alongside other tests, peroxisomal metabolites were measured in plasma and erythrocytes. The levels of VLCFAs, the branched-chain fatty acids phytanic acid and pristanic acid and the C 27 -bile acid intermediates were all elevated in plasma (Table 1), with the exception of the branched-chain fatty acid levels in patients 1, 5 and 6, which were normal. The levels of plasmalogens in erythrocytes were normal in all examined patients. Studies in cultured skin fibroblasts (Table 2) revealed increased levels of VLCFAs with a marginally decreased β-oxidation rate of C26:0 in patient 3-6. All other parameters (phytanic acid α-oxidation, pristanic acid β-oxidation and the activity of DHAPAT, the first enzyme of the etherphospholipid biosynthesis pathway) were within the control range. In addition, immunoblot analysis showed that the peroxisomal enzymes AOXI, DBP and thiolase I were normally processed (supplemental Figure S1). Immunofluorescence microscopy analysis using antibodies raised against catalase, a peroxisomal matrix enzyme, and antibodies against ALD protein, a peroxisomal membrane protein, revealed the presence of peroxisomes. However, the peroxisomes were markedly enlarged in size and reduced in number when compared to control fibroblasts (Figure 2). The presence of enlarged peroxisomes and normal immunoblot profiles, in combination with normal plasmalogens levels and DHAPAT activity usually points to a single peroxisomal enzyme deficiency (i.e. AOXI or DBP deficiency). However, measurement of the activities of AOXI and DBP in the patient cells revealed no abnormalities (Table 2). Based on these results a novel variant of a PBD with enlarged peroxisomes was suspected. Mutation analysis To determine whether the patients had a novel variant of a PBD due to mutations in 83

86 Chapter 5 5 Figure 2. Catalase Immunofluorescence in cultured fibroblasts. Catalase immunofluorescence in fibroblasts from a control subject (A), a patient with classical Zellweger Syndrome (B), a patient with peroxisomal acyl-coa oxidase I deficiency (C), and patient 3 (D). The phenotype observed in patient 3 with a reduced number of enlarged import-competent peroxisomes was similar to that observed in all five examined patients. any of the currently known 12 PEX genes, we sequenced all 12 PEX genes either in genomic DNA or in cdna prepared from the corresponding mrnas. Unexpectedly, we identified five novel apparent homozygous mutations in the PEX16 cdnas (Table 3). The mutations were also checked and appeared homozygous in genomic DNA. The PEX16 gene is localized at chromosome 11p12-p11.2 and consists of 11 exons. In humans, two different mrna variants of PEX16 are produced as a result of alternative splicing, each with an alternate exon 11 (exon 11a and exon 11b). Both transcription variants are expressed in human fibroblasts, of which variant 1 containing exon 11a is most abundant. We identified two missense mutations, two small deletions and a large genomic deletion of exon 11a, which are all located in the carboxy-terminal end of PEX16. The c.984delg in exon 11a in patient 1 and 2 results in a frame shift and introduces a termination codon at amino acid position 357. Exon 11b is unaffected in patient 1 and 2. The small deletion in patient 3 results in a Table 3. PEX16 mutations identified. Mutations Nucleotide Amino acid Exon Patient c.984delg p.i330sfsx27 11 Patient 3 c.753_755deltgt p.v252del 8 Patient 4 c.865c>a p.p289t 9 Patient 5 c.992a>g p.y331c 11 Patient 6 c _ Reference sequence of PEX16: GenBank accession number NM_ Nucleotide numbering starts at the adenine of the translation initiation codon ATG. 84

87 Identification of an unusual Variant PBD deletion of a valine at position 252. Patients 4 and 5 both have a missense mutation, leading to the amino acid substitution of a proline to a threonine at position 289 (patient 4) and of a tyrosine to a cysteine at position 331 (patient 5), respectively. The c.992a>g (p.y331c) mutation in patient 5 is located in exon 11a. Patient 6 has a large intragenic deletion in transcription variant 1 comprising the last 468 base pairs of intron 10, the entire exon 11a and the first 80 base pairs of the 3 flanking region of exon 11a and in transcription variant 2 comprising the last 603 base pairs of intron 10, and the first 4 base pairs of exon 11b. Investigation of the effect of this deletion on cdna revealed 3 splice products encoding the following amino acid sequences p.r318sfsx138, p.r318ifsx38 and p.e296dfsx33. 5 Figure 3. Catalase Immunofluorescence and complementation studies in fibroblasts. Catalase immunofluorescence in a control cell line (A), patient cell line 4 (B) and patient cell line 4 transfected with PEX16 cdna (C) and PEX12 cdna (D). Expression of wild type PEX16 cdna resulted in restoration of number and size of peroxisomes in the different patient cell lines. Three mutated PEX16 cdna constructs harboring the mutations identified in patients 1-4 (PEX16_753delTGT (F), PEX16_865C>A (G) and PEX16_984delG (H) were expressed in a complete PEX16-deficient cell line and compared with expression of wild type PEX16 cdna (E). Restoration of peroxisomes was visualized by means of catalase immunofluorescence 4 days after transfection. Expression of wild type PEX16 cdna revealed full restoration of peroxisome formation in more than 95% of the transfected cells. Expression of the mutated PEX16 cdnas revealed enlarged peroxisomes in approximately 30% of the transfected cells. 85

88 Chapter 5 5 Complementation assays To confirm that the identified mutations in PEX16 are the underlying cause of the peroxisomal abnormalities observed in fibroblasts of the patients, the cell lines were transfected with wild type PEX16 cdna, and PEX12 cdna as a negative control. Expression of wild type PEX16 cdna in the cell lines of the unrelated patients (2-6) restored the number of peroxisomes to the number found in control fibroblasts and also reduced the size of peroxisomes to normal (Figure 3C). In contrast, expression of wild type PEX12 cdna did not complement the peroxisomal abnormalities in the patient cell lines (Figure 3D). In addition, a fibroblast cell line with a complete PEX16-deficiency, resulting in the total absence of peroxisomal structures, was transfected with wild type PEX16 cdna, and the mutant PEX16 cdnas of patients 1-4. Expression of wild type PEX16 cdna resulted in more than normal sized peroxisomes in nearly all (120 of the 130) transfected cells (Figure 3E). However, expression of the PEX16 cdnas containing either the c.865c>a, the c.984delg or the c.753_755deltgt mutation, resulted in approximately enlarged peroxisomes in approximately 30% of the transfected cells while in the remaining 70% of the transfected cells no restoration of peroxisomes was found. Discussion In the present study, we identified 6 patients including one sib pair with different defects in PEX16 and an unexpected phenotype in skin fibroblasts. PEX16 was previously shown to be involved in peroxisomal membrane protein import and in line with this role; no peroxisomal remnants and a complete lack of peroxisomal functions were found in fibroblasts of the 3 PEX16-deficient patients reported in literature to date (Honsho et al., 1998; Shimozawa et al., 2002). Despite the extensive diagnostic work up in patient 1-4 no definite diagnosis could be made for a long time because the phenotypic presentation was highly unusual for a PBD. The biochemical parameters in plasma of these patients showed elevated levels of VLCFAs, phytanic acid, pristanic acid and the C 27 -bile acid intermediates but normal plasmalogen levels in erythrocytes. Moreover, in fibroblasts catalase and ALDP immunofluorescence revealed enlarged, import-competent peroxisomes which were reduced in number (Figure 2). This phenotype is typical for peroxisomal single enzyme deficiencies, i.e. AOXI but especially DBP deficiency. However, AOXI and DBP activities were completely normal in these patients. Instead, a defect in PEX16 was identified (Table 3). The identification of the PEX16 defect in patients 1-4 allowed a rapid diagnosis for patient 5 and 6. The fibroblasts of patients 5 and 6 were sent only recently to our laboratory for diagnostic work up. Because the results of the biochemical tests in their fibroblasts were very similar to the results obtained for patients 1-4, we suspected a possible defect in PEX16. Subsequent mutation analysis indeed revealed mutations in the PEX16 gene of these patients. This shows that the biochemical phenotype in fibroblasts is consistent. The biochemical presentation in plasma is also similar for all examined patients, although there is some variability in the level of abnormality for both branched-chain fatty acids and bile acid intermediates. 86

89 Identification of an unusual Variant PBD PEX16 contains two transmembrane domains (TMDs), consisting of amino acids and amino acids Both the amino- and carboxy-terminal ends are exposed into the cytosol (South and Gould, 1999). Amino acids are needed for binding to PEX19 and for sorting to the peroxisomal membrane (Fransen et al., 2001). The mutations which we identified, two missense mutations and 3 deletions, appear to have a mild effect on the function of PEX16 and are not located in one of the known functional domains of PEX16, but are all located in the carboxy-terminal end of PEX16. The detected changes have not been reported previously as either mutations or polymorphisms in the NCBI SNP database. To demonstrate that the mutations we identified cause the phenotype observed in fibroblasts of these patients, we transfected the patient cell lines with wild type PEX16 cdna (transcript variant 1) and studied whether complementation occurred. In addition, the effect of the mutated PEX16 cdnas of patients 1-4 on peroxisomal size and number was studied. These experiments showed that the mutant PEX16 proteins are functional, but not to the same extent as wild type PEX16. The expression of the wild-type PEX16 restored biogenesis of peroxisomes in nearly all cells whereas expression of the mutant PEX16 cdnas resulted in peroxisomes in approximately 30% of the transfected cells. Possibly, the concentration needed to restore the biogenesis of peroxisomes in all cells was not reached when using the mutated PEX16 cdnas for transfection due to a reduced stability of the mutant proteins. Interestingly, two transcription variants of PEX16 exist containing either exon 11a or exon 11b. Three homozygous mutations were identified in exon 11 of which two only affected the transcript variant 1 with exon 11a (patient 1, 2 and 5) and one affected both transcript variants (patient 6). The phenotype in fibroblasts was undistinguishable for these different mutations, showing that mutations in transcript variant 1 cause the phenotype of enlarged import-competent peroxisomes. The phenotype of a small number of enlarged peroxisomes caused by the identified mutations in the PEX16 gene suggests that PEX16 is involved in the morphology and division of peroxisomes in addition to its involvement in membrane assembly. 5 The peroxisomes in the patients fibroblasts are able to import matrix and membrane proteins, and this import is sufficient to keep the α- and β-oxidation rate at normal levels (Table 2). However, it could very well be that in other organs such as the liver, peroxisomal functions are more severely affected since the patients did accumulate peroxisomal metabolites in plasma. This hypothesis is supported by studies in a liver biopsy from patient 3 which showed parenchymal cells with peroxisomes devoid of the matrix enzymes catalase and alanine-glyoxylate aminotransferase (Pineda et al., 1999). The observed MRI appearances of progressive leukodystrophy and selective brain atrophy merits some discussion. MR signal characteristics of abnormalities that are greater on T2 than T1 weighted images have been previously described as a hypomyelination pattern of leukodystrophy (Schiffmann and van der Knaap, 2009); however, these changes were seen developing in white matter that had previously appeared normally myelinated on MRI. The observation of selective atrophy of the corpus callosum and cerebellum in combination with this pattern of progressive leukodystrophy is judged to be unique. 87

90 Chapter 5 In summary, our results show that in cases with only very mild biochemical peroxisomal abnormalities in fibroblasts, a PBD should not be excluded when peroxisomal metabolites in plasma are abnormal. Moreover, although PEX16 is involved in peroxisomal membrane assembly, PEX16 defects can present with import-competent peroxisomes in fibroblasts. This is important for future diagnostics of patients with a peroxisomal disorder. Acknowledgement This study was supported by a grant from the Prinses Beatrix Fonds (MAR03_0216), the FP6 European Union Project peroxisomes (LSHG-CT ) and a grant from the Netherlands Organization for Scientific research (NWO grant ). We thank Dr. M. Pineda and Dr. M. Giros for referring their patients to us and we thank the families for their permission to publish this article. 5 References Dacremont G, Vincent G Assay of plasmalogens and polyunsaturated fatty acids (PUFA) in erythrocytes and fibroblasts. J Inherit Metab Dis 18 Suppl 1: Dacremont G, Cocquyt G, Vincent G Measurement of very long-chain fatty acids, phytanic and pristanic acid in plasma and cultured fibroblasts by gas chromatography. J Inherit Metab Dis 18 Suppl 1: Fransen M, Wylin T, Brees C, Mannaerts GP, Van Veldhoven PP Human pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. Mol Cell Biol 21: Gould SJ, Raymond GV, Valle D The peroxisome biogenesis disorders. In: Scriver C.R., Beaudet A.L., Sly W.S., Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill, Inc. p Honsho M, Tamura S, Shimozawa N, Suzuki Y, Kondo N, Fujiki Y Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D. Am J Hum Genet 63: Ofman R, Wanders RJ Purification of peroxisomal acyl-coa: dihydroxyacetonephosphate acyltransferase from human placenta. Biochim Biophys Acta 1206: Pineda M, Giros M, Roels F, Espeel M, Ruiz M, Moser A, Moser HW, Wanders RJ, Pavia C, Conill J, Aracil A, Amat L, Pampols T Diagnosis and follow-up of a case of peroxisomal disorder with peroxisomal mosaicism. J Child Neurol 14: Poll-The BT, Saudubray JM, Ogier HA, Odievre M, Scotto JM, Monnens L, Govaerts LC, Roels F, Cornelis A, Schutgens RB, Infantile Refsum disease: an inherited peroxisomal disorder. Comparison with Zellweger syndrome and neonatal adrenoleukodystrophy. Eur J Pediatr 146: Schiffmann R, van der Knaap MS Invited article: an MRI-based approach to the diagnosis of white matter disorders. Neurology 72: Shimozawa N, Suzuki Y, Zhang Z, Imamura A, Ghaedi K, Fujiki Y, Kondo N Identification of PEX3 as the gene mutated in a Zellweger syndrome patient lacking peroxisomal remnant structures. Hum Mol Genet 9: Shimozawa N, Nagase T, Takemoto Y, Suzuki Y, Fujiki Y, Wanders RJ, Kondo N A novel aberrant splicing mutation of the PEX16 gene in two patients with Zellweger syndrome. Biochem Biophys Res Commun 292: South ST, Gould SJ Peroxisome synthesis in the absence of preexisting peroxisomes. J Cell Biol 144: van Grunsven EG, van Berkel E, Mooijer PA, Watkins PA, Moser HW, Suzuki Y, Jiang LL, Hashimoto T, Hoefler G, Adamski J, Wanders RJ Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis. Am J Hum Genet 64:

91 Identification of an unusual Variant PBD Vreken P, van Lint AE, Bootsma AH, Overmars H, Wanders RJ, van Gennip AH Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry. J Chromatogr B Biomed Sci Appl 713: Wanders BJ, Denis SW, Dacremont G Studies on the substrate specificity of the inducible and noninducible acyl-coa oxidases from rat kidney peroxisomes. J Biochem 113: Wanders RJ, van Roermund CW Studies on phytanic acid alpha-oxidation in rat liver and cultured human skin fibroblasts. Biochim Biophys Acta 1167: Wanders RJ, Dekker C, Ofman R, Schutgens RB, Mooijer P. 1995a. Immunoblot analysis of peroxisomal proteins in liver and fibroblasts from patients. J Inherit Metab Dis 18 Suppl 1: Wanders RJ, Denis S, Ruiter JP, Schutgens RB, van Roermund CW, Jacobs BS. 1995b. Measurement of peroxisomal fatty acid beta-oxidation in cultured human skin fibroblasts. J Inherit Metab Dis 18 Suppl 1: Wanders RJ, Waterham HR Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders. Clin Genet 67:

92 Chapter 5 Supplemental Figure S1. Peroxisomal processing. Immunoblot analysis of the peroxisomal matrix proteins AOXI (A), thiolase I (B) and DBP (C) in fibroblasts homogenates from a control subject (lane 1), patient 1 (lane 2), patient 3 (lane 3), patient 4 (lane 4), patient 5 (lane 5), patient 6 (lane 6) and a ZS patient (lane 7). The molecular masses of the proteins in kda have been indicated (arrows). 5 Supplemental Table 1. Primer sets used for PEX16 mutation analysis. Amplicon Primers Exon of gdna Regions of cdna A [-21M13]-GAAGCAGGAAGGAGGGCG 1 and 2 [M13-Rev]-ATTCAGTCATAGCACAAGGTG B [-21M13]-TGTGAGATCATGTTGGGGAG 3 [M13-Rev]-CTAAGATGGGAATACTCACAC C [-21M13]-GTCAGAGAAGCTCCCTCCTAG 4 and 5 [M13-Rev]-TACTGTATTCATGCTGGTTGG D [-21M13]-CCTGCTTGTAGTTCCCTTGAC 6, 7 and 8 [M13-Rev]-ATTATAGCAGAAAGCCCAGTG E [-21M13]-ACATAGGCGGGGTGGCAG 9 [M13-Rev]-CCCGGACAACACACAGTGC F [-21M13]-GCACGGTGGTCAGTGAAGG 10 and 11 [M13-Rev]-TATGGCTGCCGAGGCGAG 1 [-21M13]-TGTCGGTGCCGAGGGCAGGAT c.1-19_352 [M13-Rev]-AGCTGGATGAGGGCGATGACA 2 [-21M13]-TGTTCATGGAGATGGGAGCT c.281_721 [M13-Rev]-AAGAGCCAGGGTTTCCACGA 3 [-21M13]-TTTGTACATTGCCCGGCCGCT c.666_ [M13-Rev]-AGGGAGCCCCTCTTCCCTAAT 90

93 Chapter 6 Mutations in PEX10 are a Cause of Autosomal Recessive Ataxia Luc Régal 1, Merel S Ebberink 2, Nathalie Goemans 3, Ronald JA Wanders 2, Linda De Meirleir 4, Jacques Jaeken 1, Maarten Schrooten 5, Rudy Van Coster 4, Hans R Waterham 2 1 Department of Pediatrics, Metabolic Center, University Hospital Leuven, Belgium; 2 Laboratory Genetic Metabolic Diseases, Academic Medical Center Amsterdam, the Netherlands; 3 Department of Pediatrics, Child Neurology, University Hospital Leuven, Belgium; 4 Department of Pediatric Neurology and Metabolic Diseases, University Hospital Ghent, Belgium; 5 Department of Neurology, University Hospital Leuven, Belgium. Annals of Neurology (2010)

94 Chapter 6 Abstract Peroxisome biogenesis disorders typically cause severe multi-system disease and early death. We describe a child and an adult of normal intelligence with progressive ataxia, axonal motor neuropathy and decreased vibration sense. Both patients had marked cerebellar atrophy. Peroxisomal studies revealed a peroxisome biogenesis disorder. Two mutations in PEX10 were found in the child: c.992g>a (novel) and c.764_765insa, and in the adult: c.2t>c (novel) and c.790c>t. Transfection with wild type PEX10 cdna corrected the fibroblast phenotype. Bile acid supplements and dietary restriction of phytanic acid were started. Peroxisome biogenesis disorders should be considered in the differential diagnosis of autosomal recessive ataxia. 6 92

95 Mutations in PEX10 are a Cause of Autosomal Recessive Ataxia Introduction Generalized defects in peroxisome biogenesis (PBD) cause Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD), in order of decreasing severity. They are collectively known as the Zellweger spectrum disorders. Mutations in 12 different PEX genes have been associated with the Zellweger spectrum. This spectrum is characterized clinically by severe global neurological involvement and a variable severity of dysmorphism, retinitis pigmentosa, sensorineural deafness, liver disease and other systemic features, including death in infancy or early childhood. Metabolites in blood show evidence of generalized peroxisomal dysfunction (Steinberg et al., 2006). More recently, patients with milder presentations of PBD have been identified. We report two unrelated patients of normal intelligence with cerebellar atrophy, slowly progressive ataxia, axonal motor neuropathy and posterior column dysfunction, caused by a PBD. In both patients, mutations in PEX10 were found. Case reports Patient 1 was referred at the age of 8.5 years because of worsening balance problems, appearing at age 5 years. He had a history of neonatal jaundice. Early development was normal, he walked at age 18 months and his growth followed the 75th percentile. He follows normal education. Clinical examination revealed cerebellar signs with gait ataxia, limb ataxia, mild cerebellar dysarthria and impaired smooth pursuit. Ankle tendon reflexes were absent, and vibration sense was 6/8 on a graded tuning fork in the lower limbs, later deteriorating to 4/8 in the lower limbs and 6/8 in the upper limbs. The cerebellum was atrophic without white matter or spinal cord involvement on magnetic resonance imaging (MRI) and H1 MR spectroscopy confirmed neuronal loss (Figure 1). EMG and nerve conduction 6 Figure 1. Cerebral MRI and MRS. A-D: patient 1, cerebellar atrophy on sagittal (A) and axial (C, D) T2-weighted images, with normal white matter and the decreased N-acetyl-aspartate peak (arrow) in the cerebellum on H1MRS (B). E-H: patient 2, cerebellar atrophy at age 8 years (E), and MRI at age 19 years. 93

96 Chapter 6 studies indicated an axonal motor neuropathy. The latency of N20 was prolonged on median nerve SSEP, corresponding with the clinical posterior column involvement. Electroretinogram, ophthalmoscopy and abdominal sonography were normal. Liver function tests were mildly abnormal. Total serum cholesterol was 65 mg/dl ( ). Serum very-long-chain fatty acids (VLCFA) levels were within the normal range, but phytanic and pristanic acid levels were clearly increased, as were bile acid intermediates and pipecolic acid. Plasmalogens in erythrocytes were normal (Table 1). Other investigations, including vitamin E levels, α-fetoprotein, lactate in plasma and cerebrospinal fluid, plasma amino acids, urinary organic acids, 7- and 8-dehydrocholesterol, karyotype, analysis for trinucleotide expansions in FRDA1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, ATXN8OS, and PPP2R2B, and Table 1. Biochemical parameters in plasma, erythrocytes and cultured skin fibroblasts. 6 Peroxisomal test Patient 1 Patient 2 Control Zellweger Plasma C26:0 (µmol/l) C26/C C24/C Pristanic acid (µmol/l) Phytanic acid (µmol/l) Bile acid intermediated - THCA (µmol/l) DHCA (µmol/l) Pipecolic acid (µmol/l) Erythrocytes Plasmalogens - C16:0 dma/c16:0 - C18:0 dma/c18: ± ±0.056 Fibroblasts VLCFAs - C22:0 (µmol/g protein) abnormal - C24:0 (µmol/g protein) - C26:0 (µmol/g protein) - C26/C α-oxidation - phytanic acid (pmol/h.mg) ± β-oxidation - C26:0 (pmol/h.mg) ± pristanic acid (pmol/h.mg) ± DHAPAT activity (nmol/2h.mg) Thiolase immunoblot - 41 kda - 44 kda ACOX immunoblot - 72 kda - 52 kda - 20kDa THCA, trihydroxycholestanoic acid; DHCA, dihydroxycholestanoic acid; VLCFA, very-long-chain fatty acid; DHAPAT, dihydroxy-acetone phosphate acyltransferase; ACOX, acyl-coenzyme A oxidase; dma, dimethyl acetal. Control values are 5%-95% ranges or mean ± SD. 94

97 Mutations in PEX10 are a Cause of Autosomal Recessive Ataxia pentanucleotide expansions in ATXN10, were normal. Because of the progressive ataxia and elevated phytanic acid levels, a diet low in phytanic acid was started at age 9.5 years. This lowered plasma levels of phytanic acid to 2.9 µmol/l and pristanic acid to 4.5 µmol/l. While ataxia, as measured with the scale for the assessment and rating of ataxia (SARA; Schmitz-Hubsch et al., 2006), worsened from 9 to 17 in the year before introduction of the diet, it has stabilized in the year thereafter, and SARA score at the age of 11 year was 14. After informed consent, a skin biopsy was taken and fibroblasts were cultured for peroxisomal analyses. Patient 2 presented at the age of 6 years with slowly progressive gait problems. Clinical examination showed cerebellar ataxia and weak tendon reflexes. In the following years, there was a slow neurological deterioration, and the patient developed areflexia, pes cavus, atrophy of distal muscles and decreased vibration sense. At age 21 years, he was still ambulatory without support, scored 15 on the SARA and his vibration sense in the lower limbs was 2/8 on a graded tuning fork. EMG showed axonal motor neuropathy. Somatosensory evoked potentials were consistent with posterior column dysfunction. MRI of the brain showed cerebellar atrophy, which was not clearly progressive (Figure 1), but no white matter involvement. Blood chemistry showed low cholesterol and mild elevation of CK. Absolute VLCFA levels were not elevated, but C26:0/C24:0 and C26:0/C22:0 ratios and phytanic acid were mildly elevated. As the phenotype reminded us of patient 1, other peroxisomal metabolites were measured, showing increases in pristanic acid, bile acid intermediates and pipecolic acid, and low plasmalogens. These results were consistent with a PBD (Table 1). After informed consent, a skin biopsy was taken and peroxisomal analyses were performed on cultured skin fibroblasts. A diet restricted in phytanic acid was started at age 20.5 years. However, levels of phytanic acid and pristanic acid remained elevated, possibly due to compliance issues. Ursodeoxycholic acid, vitamin D, and vitamin E were supplemented. Bile acid intermediates normalized, and ataxia, measured with the SARA remained stable at 15 in the year after introduction of therapy. 6 Methods VLCFA levels, peroxisomal beta-oxidation, alpha-oxidation and dihydroxyacetonephosphate acyltransferase (DHAPAT) activity were measured in cultured fibroblasts (Gootjes et al., 2004). Immunoblot analysis for acyl-coa oxidase and the peroxisomal thiolase, and immunofluorescence for catalase at 37 C and 40 C was done as described (Gootjes et al., 2004). PEX12 (Gootjes et al., 2004), PEX2 (Shimozawa et al., 2000), and PEX10 (Krause et al., 2006) were sequenced. Cultured fibroblasts were transfected with PEX10 cdna using the AMAXA nucleofector technology (Gootjes et al., 2004). Nucleotides in PEX10 are numbered from the first ATG according to the cdna sequence of GenBank accession number NM_ Accordingly, c.764_765insa corresponds to c.704_705insa and c.790c>t corresponds to c.730c>t in earlier publications (Krause et al., 2006). 95

98 Chapter 6 A B C D Figure 2: Catalase immunofluorescence studies (A-C) and PEX co-transfection studies (D-E) in cultured fibroblasts. A: control fibroblasts showing punctuated immunofluoresent pattern, corresponding to catalase importcompetent peroxisomes. B: fibroblasts from patient 1 cultured at 40 C, showing catalase distribution in most of the cells (peroxisomal) but not in others (nuclear and cytoplasmic immunofluorescence for catalase), i.e. peroxisomal mosaicism. D and E: fibroblasts of patient 1 co-transfected with either PEX10 cdna (D) or PEX13 cdna (E) and GFP-SKL. Only overexpression of PEX10 cdna restores normal peroxisomal biogenesis in transfected cells. 6 Results In the fibroblasts of patient 1, peroxisomal alpha- and beta-oxidation and DHAPAT activity in fibroblasts were normal. In the fibroblasts of patient 2, C26:0/C22:0 was mildly elevated and pristanic acid beta oxidation was low. Immunofluorescence for catalase showed peroxisomes in approximatley 80% of the cultured fibroblasts. This peroxisomal mosaicism was still observed at 40 C (Figure 2), making genetic complementation analysis based on restoration of peroxisome biogenesis to identify the defective PEX gene problematic. Consequently, it was decided to sequence candidate PEX genes (Gootjes et al., 2004). Sequence analysis of the PEX2 and PEX12 genes in patient 1 did not reveal mutations. However, two heterozygous mutations were found in the PEX10 gene. One mutation, c.764_765insa, was previously reported in patients with Zellweger syndrome (Warren et al., 2000; Steinberg et al., 2004; Turner et al., 2007). The second mutation, c.992g>a, changes a highly conserved arginine at position 331, the last amino acid of the crucial RING finger domain (Warren et al., 2000), into a glutamine. This mutation has not been described previously. The latter mutation is most probably responsible for the mild clinical presentation. In patient 2 also two different heterozygous mutations were found in PEX10. The first mutation, c.2t>c, abolishes the translation initiation codon. If translation would start at the next in-frame methionine at position 145, the resulting protein would lack the first transmembrane domain. This mutation has not been described before. The second mutation, c.790c>t, replaces arginine at position 264 by a stop codon, resulting in a protein truncated upstream of the RING finger domain (Krause et al., 2006). The parents were each heterozygous for one of the mutations, confirming compound heterozygosity in the patient. PEX10 was confirmed to be defective in both patients by transfection of the patient fibroblasts with wild-type PEX10 cdna, which resulted in normal peroxisome 96

99 Mutations in PEX10 are a Cause of Autosomal Recessive Ataxia formation in nearly all transfected cells (Figure 2). In contrast, expression of PEX13 cdna did not result in normal peroxisome formation. Discussion We describe two patients with a strikingly similar biochemical and clinical phenotype caused by mutations in PEX10. Each patient was heterozygous for a known, severe, mutation and a novel, apparently milder, mutation. Biochemically, their absolute levels of VLCFA in blood were normal. The mildly increased ratio of C26:0/C22:0 without elevation of C26:0 observed in our patients is usually not considered indicative of peroxisomal disease. In a generalized PBD, absolute VLCFA levels are usually clearly elevated. Both patients had elevated plasma levels of phytanic acid, pristanic acid, bile acid intermediates, and pipecolic acid suggesting a generalized PBD, which was supported by the decreased erythrocyte plasmalogens in patient 2. Remarkably, however, apart from a slight elevation of C26:0/C22:0 ratio in patient 2 and low pristanic acid oxidation in patient 2, peroxisomal biochemical studies in cultured fibroblasts were normal, in contrast to what typically is observed in generalized PBD (Table 1). Peroxisomal mosaicism, i.e. a mixed population of fibroblasts with and without peroxisomes, has been described previously in other patients with milder mutations in other PEX genes. This mosaicism makes genetic complementation analysis to identify the defective PEX gene problematic. Culturing fibroblasts at 40 C has been used in patients with mosaicism to make all fibroblasts deficient in peroxisomes and thus eliminate mosaicism (Gootjes et al., 2004), but in our patients the mosaicism persisted at 40 C. Systematic sequencing of the different PEX genes was therefore necessary. The combination of cerebellar ataxia, axonal motor neuropathy and posterior column dysfunction without mental retardation was virtually identical in both patients, while the mutations in PEX10 were different. The peroxisomal disease in these patients is very mild in comparison with other published patients with mutations in PEX10, who belonged to the severe ZS and NALD subgroups of the Zellweger spectrum ( Interestingly, since our first presentation of ataxia caused by mutations in PEX10 (Régal et al., 2008b; Régal et al., 2008a), a previously reported patient (Clayton et al., 1996) with a similar phenotype, but with cerebral white matter changes, was recently reported also to have mutations in PEX10 (Steinberg et al., 2009). This suggests that mild mutations in PEX10 are a novel cause of autosomal recessive ataxia. Other patients with comparable clinical and biochemical features in whom PEX10 deficiency was not evaluated have been reported (MacCollin et al., 1990; Roels et al., 2003). As conditional knock-out of PEX5 in the liver but not in brain resulted in persistent cerebellar atrophy in mice (Krysko et al., 2007). The cerebellar atrophy in our patients may represent a secondary effect of circulating metabolites. This hypothesis is supported by the apparent stabilization with therapy influencing these metabolites in our patients. Most recently, mutations in D-amino acid oxidase, a peroxisomal enzyme, were found in familial amyotrophic lateral sclerosis (De Belleroche et al., 2009). D-amino acid oxidase is responsible for the degradation of among others, 6 97

100 Chapter 6 D-serine, an NMDA agonist. Deficiency of D-amino acid oxidase might therefore result in excitotoxicity. Whether the motor neuron involvement in our patients is related to secondary dysfunction of D-amino acid oxidase remains to be evaluated. Mild mutations in other PEX genes have also been associated with peroxisomal mosaicism. Patients with peroxisomal mosaicism and PEX6 mutations have retinopathy and hearing loss, but no ataxia, cerebellar atrophy or neuropathy (Raas- Rothschild et al., 2002), personal observation). Mild PEX12 mutations associated with peroxisomal mosaicism are reported to cause mental retardation and hearing loss, without mention of ataxia (Gootjes et al., 2004). This suggests that the clinical consequences of mild mutations in different PEX genes may be gene-specific, at least to some degree, in contrast to the unspecific severe consequences of the severe mutations. Both our patients were heterozygous for 1 severe mutation. It can be expected that patients carrying two mild mutations will have even milder clinical presentations. Searching for mild PBD in patients of different ages with unexplained ataxia may therefore be worthwhile, particularly as this disease appears to be partially treatable. 6 References Clayton PT, Johnson AW, Mills KA, Lynes GW, Wilson J, Casteels M, Mannaerts G Ataxia associated with increased plasma concentrations of pristanic acid, phytanic acid and C27 bile acids but normal fibroblast branched-chain fatty acid oxidation. J Inherit Metab Dis 19: De Belleroche J, Mitchell J, Praveen P A novel putative familial ALS locus on chromosome 12: D-amino acid oxidase. Gootjes J, Schmohl F, Mooijer PA, Dekker C, Mandel H, Topcu M, Huemer M, Von SM, Marquardt T, Smeitink JA, Waterham HR, Wanders RJ Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism. Hum Mutat 24: Krause C, Rosewich H, Thanos M, Gartner J Identification of novel mutations in PEX2, PEX6, PEX10, PEX12, and PEX13 in Zellweger spectrum patients. Hum Mutat 27:1157. Krysko O, Hulshagen L, Janssen A, Schutz G, Klein R, De BM, Espeel M, Gressens P, Baes M Neocortical and cerebellar developmental abnormalities in conditions of selective elimination of peroxisomes from brain or from liver. J Neurosci Res 85: MacCollin M, De Vivo DC, Moser AB, Beard M Ataxia and peripheral neuropathy: a benign variant of peroxisome dysgenesis. Ann Neurol 28: Raas-Rothschild A, Wanders RJ, Mooijer PA, Gootjes J, Waterham HR, Gutman A, Suzuki Y, Shimozawa N, Kondo N, Eshel G, Espeel M, Roels F, Korman SH A PEX6-defective peroxisomal biogenesis disorder with severe phenotype in an infant, versus mild phenotype resembling Usher syndrome in the affected parents. Am J Hum Genet 70: Régal L, Ebberink MS, Goemans N, Wanders RJA, Waterham HR, Jaelen J. 2008a. A peroxisomal biogenesis disorder causing spinocerebellar ataxia in an adult. Régal L, Ebberink MS, Wanders RJA, Wuyts B, Waterham HR, van Driessche M, van Coster R, Achten E, de Meirleir L. 2008b. A peroxisomal form of spinocerebellar ataxia caused by mutations in PEX10. Roels F, Baes M, de Bie S Peroxisomal disorders and regulation of genes. New York: Kluwer Academic/Plenum Publisher. Schmitz-Hubsch T, du Montcel ST, Baliko L, Berciano J, Boesch S, Depondt C, Giunti P, Globas C, Infante J, Kang JS, Kremer B, Mariotti C, Melegh B, Pandolfo M, Rakowicz M, Ribai P, Rola R, Schols L, Szymanski S, van de Warrenburg BP, Durr A, Klockgether T, Fancellu R Scale for the assessment and rating of ataxia: development of a new clinical scale. Neurology 66: Shimozawa N, Zhang Z, Imamura A, Suzuki Y, Fujiki Y, Tsukamoto T, Osumi T, Aubourg P, Wanders RJ, Kondo N Molecular mechanism of detectable catalase-containing particles, peroxisomes, in 98

101 Mutations in PEX10 are a Cause of Autosomal Recessive Ataxia fibroblasts from a PEX2-defective patient. Biochem Biophys Res Commun 268: Steinberg S, Chen L, Wei L, Moser A, Moser H, Cutting G, Braverman N The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum. Mol Genet Metab 83: Steinberg SJ, Dodt G, Raymond GV, Braverman NE, Moser AB, Moser HW Peroxisome biogenesis disorders. Biochim Biophys Acta 1763: Steinberg SJ, Snowden A, Braverman NE, Chen L, Watkins PA, Clayton PT, Setchell KD, Heubi JE, Raymond GV, Moser AB, Moser HW A PEX10 defect in a patient with no detectable defect in peroxisome assembly or metabolism in cultured fibroblasts. J Inherit Metab Dis 32: Turner CL, Bunyan DJ, Thomas NS, Mackay DJ, Jones HP, Waterham HR, Wanders RJ, Temple IK Zellweger syndrome resulting from maternal isodisomy of chromosome 1. Am J Med Genet A 143A: Warren DS, Wolfe BD, Gould SJ Phenotype-genotype relationships in PEX10-deficient peroxisome biogenesis disorder patients. Hum Mutat 15:

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103 Chapter 7 A novel human peroxisome biogenesis disorder affecting peroxisome division Ebberink MS 1, Koster J 1, Stolte-Dijkstra I 2, Visser G 3, van Spoonsen FJ 4, Smit GPA 5, Mooijer PAW 1, Wanders RJA 1, Waterham HR 1 1 Academic Medical Center, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Departments of Paediatrics/Emma Children s Hospital, Amsterdam, The Netherlands; 2 Department of Clinical Genetics, University Medical Center Groningen, University of Groningen, The Netherlands; 3 Metabolic Diseases, Wilhelmina Children s Hospital, Utrecht, The Netherlands; 4 Beatrix Children s Hospital, University Medical Center of Groningen, University of Groningen, Groningen, The Netherlands; 5 Beatrix Children s Hospital, University Medical Center of Groningen, University of Groningen, Department of Metabolic Dieseases, Groningen, The Netherlands In preparation for submission

104 Chapter 7 Abstract 7 Objective Peroxisomes are dynamic organelles that divide continuously and adjust their protein content in response to the metabolic need. In humans, currently 16 different peroxins (PEX), encoded by PEX genes, have been implicated in various stages of peroxisome biogenesis, protein import, and division. The division of peroxisomes involves elongation of existing peroxisomes followed by membrane constriction and finally fission of peroxisomal tubules. PEX11 has been implicated in the step of peroxisome elongation. In mammals, three PEX11 isoforms (PEX11α, PEX11β and PEX11γ) have been identified. Here, we report the identification of the first patient with a defect in PEX11β. Results The male patient displayed clinical features of the Zellweger syndrome spectrum disorders but, except for one single occasion, showed no peroxisomal biochemical abnormalities in plasma, erythrocytes and cultured fibroblasts. However, the peroxisomal appearance in fibroblasts of the patient was abnormal, indicative of a defect in peroxisome division. After excluding mutations in any of the 12 PEX genes currently implicated in Zellweger syndrome spectrum disorders, we identified a homozygous nonsense mutation c.64c>t (p.q22x) in the PEX11β gene. When the patient s fibroblasts were cultured at 40 C, the majority of cells lost matrix protein-containing peroxisomes. Overexpression of PEX11β in such cells reverted the mutant phenotype to normal, while overexpression of PEX11γ resulted in partial reversion to the peroxisomal phenotype observed at 37 C. Conclusion A defect in PEX11β results in a Zellweger syndrome spectrum disorder, with virtually no peroxisomal biochemical abnormalities in plasma, erythrocytes and cultured fibroblasts, but with an abnormal peroxisomal phenotype. Our results point to a partial redundant function of PEX11γ with PEX11β, which modulates the biochemical phenotype. 102

105 A novel human peroxisome biogenesis disorder affecting peroxisome division Introduction Human peroxisomes play an important role in various essential metabolic pathways, among which the biosynthesis of ether phospholipids and the alphaand beta-oxidation of fatty acids (Wanders and Waterham, 2006). Consequently, defects in genes encoding peroxisomal proteins can lead to a variety of different peroxisomal disorders that can be categorized in two main groups, including the peroxisome biogenesis disorders (PBDs) and the single peroxisomal enzyme deficiencies (Wanders and Waterham, 2006; Weller et al., 2003). Peroxisome biogenesis disorders include the Zellweger syndrome spectrum (ZSS) disorders and Rhizomelic Chondrodysplasia Punctata type I (Weller et al., 2003). The ZSS includes three previously defined phenotypes, namely the Cerebro-Hepato-Renal Syndrome of Zellweger (i.e.zellweger Syndrome; ZS; MIM ), neonatal adrenoleukodystrophy (NALD; MIM ) and infantile Refsum disease (IRD; MIM ) with decreasing clinical and biochemical severity. Common to all three ZSS disorders are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness (Gould et al., 2001; Weller et al., 2008). In addition, patients with ZS are severely hypotonic from birth and die within their first year of life. Patients with NALD experience neonatal onset of hypotonia and seizures, have progressive white matter disease and usually die in late infancy. The survival of IRD patients is variable, with many patients surviving beyond infancy and some may even reach adulthood (Barth et al., 2001). IRD patients have no neuronal migration defect, but can develop a progressive white matter defect. 7 ZSS disorders are autosomal recessive disorders that can be caused by a defect in any of at least 12 different PEX genes (Steinberg et al., 2006). These 12 PEX genes encode proteins called peroxins and are involved in various stages of peroxisomal protein import and/or the biogenesis of peroxisomes (Steinberg et al., 2006). A number of additional peroxins and other proteins have been indentified that are involved in the proliferation and division of peroxisomes but for which no human disorders have been reported (Thoms and Erdmann, 2005). However, one of these proteins, DLP1, was recently reported to be defective in a patient with a lethal defect in the fission of mitochondria and peroxisome (Waterham et al., 2007). The division of peroxisomes occurs in distinct steps, including elongation of peroxisomes, followed by membrane constriction and finally the fission of peroxisomal tubules. PEX11 was the first peroxisomal protein to be implicated in peroxisome division (Erdmann and Blobel, 1995; Marshall et al., 1995). PEX11-deficient cells of the yeast Saccharomyces cerevisiae contain fewer and larger peroxisomes than wild-type yeast cells, while overexpression of PEX11 results in an increase in peroxisome number and the occurrence of elongated peroxisomal structures in wild-type S. cerevisiae cells (Marshall et al., 1995). In mammals, three PEX11 isoforms (PEX11α, PEX11β and PEX11γ) have been identified. They are all integral peroxisomal membrane proteins (PMPs) and have both their amino and carboxyl terminus exposed to the cytosol (Abe and Fujiki, 1998; Schrader et al., 1998). We here report the identification of the first patient with a homozygous nonsense mutation in the PEX11β gene. The patient displayed clinical features compatible with 103

106 Chapter 7 the Zellweger syndrome spectrum, but did not show clear peroxisomal biochemical abnormalities. However, microscopical studies revealed that the peroxisomes in fibroblasts of the patient were elongated and arranged in rows indicative of a defect in peroxisome division. Patient and Methods 7 Case report The index patient is a 25 years old, mentally retarded male. He is the fourth child of non consanguineous parents with a normal karyotype and has two healthy brothers and one healthy sister. The mother had three miscarriages. The birth weight was approximately 3500 grams at term. At six weeks of age, the parents noted an extreme squint and the absence of eye contact, which was caused by bilateral congenital cataracts. Investigations for congenital infections and metabolic diseases were negative. At the age of 4 months, a cataract extraction was performed. No other ophthalmological problems were noted, but his vision never has been more than 10%. He showed a normal early development and walked at the age of 1.5 years. At that age, he underwent surgery for a hydrocele of the testis. After the surgery there was a remarkable regression: he lost his speech and his ability to walk and it took half a year for this to improve. This was the only documented period of regression, although in general his development is mildly retarded. He had remarkable dry skin with scaling of the hands and feet. At the age of five years, a hearing problem was noted and this progressed to severe bilateral perceptive hearing loss with a threshold of 60 db in low frequencies to thresholds of more than 110 db from 2000 Hz onwards. Since the age of approximately 10 years, he started to complain of painful muscles and showed a gradual loss of muscle strength. At the age of 12 years, he was referred for extensive clinical and laboratory evaluation. Main features on neurological examination were a complex nystagmus with a rotatory component, normal muscle strength, low reflexes and normal sensibility in the arms, but low muscle strength, absent reflexes and disturbed sensibility in the legs. Creatine kinase was normal to slightly elevated (max. 85 U/l, normal 50 U/l). Magnetic resonance imaging of the brain revealed an Arnold Chiari malformation type I. An electroencephalogram was normal. There was no evidence of a cardiomyopathy. An electromyography showed low normal motor conduction velocity and absent sensory responses. A combined muscle/nerve biopsy showed a predominance of type I muscle fibers and a reduced number of myelinated fibers. Extensive metabolic investigations were performed. Serum lactate was 1.0 mmol/l, pyruvate: 0.6 mmol/l, urine lactate was 0.1 mmol/l. A glucose tolerance test showed no elevation of lactate. Mitochondrial oxidation velocity was slightly reduced in the muscle biopsy but the ATP and creatine phosphate production from pyruvate were within normal range. Mitochondrial enzymes, including the activities of the mitochondrial complexes in the muscle biopsy, were normal. Long chain fatty acid analyses showed normal C24:C22, C25:C22, and C26:C22 ratios, and normal phytanic acid and pipecolic acid concentrations in plasma. On one occasion a slightly elevated C26:C22 ratio of (normal < 0.026) was determined. 104

107 A novel human peroxisome biogenesis disorder affecting peroxisome division In the subsequent years, his muscle strength deteriorated and required the use of a wheel chair for longer distances. From the age of 15 years, he had regular attacks of migraines/convulsions during which he felt cold, was encephalopathic, complained of nausea and headache, did not know what he was saying or doing, did not speak correctly, had trouble with his vision and had a numb feeling in his mouth and hands. These attacks often followed upon physical or mental exertion and usually ended with violent vomiting episodes lasting a few hours to one day. Based on these episodes, a MELAS presentation or convulsions was considered and he was treated with valproic acid, co-enzyme Q10 and carnitine after which the attacks only presented once and his total condition improved. The parents provided oral informed consent for this study and publication of the results. Biochemical analysis and microscopical analysis Levels of very long chain fatty acids, phytanic and pristanic acid, and C 27 -bile acid intermediates were measured in plasma as described before (Dacremont et al., 1995). Plasmalogens were determined in erythrocytes as previously described (Dacremont and Vincent, 1995). Dihydroxyacetonephosphate acyltransferase (DHAPAT; Ofman and Wanders, 1994), concentrations of very long chain fatty acids (Vreken et al., 1998), β-oxidation of cerotic acid (C26:0), palmitic acid (C16:0) and pristanic acid (Wanders et al., 1995), and α-oxidation of phytanic acid (Wanders and van Roermund, 1993) were measured in cultured fibroblasts as previously described. Fibroblasts were cultured in DMEM medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Bio-Whittaker), 100 U/ml penicillin, 100µg/ml streptomycin and 25 mm Hepes buffer with L-glutamine in a humidified atmosphere of 5% CO 2 and at 37 C. Peroxisomes were examined by immunofluorescence microscopy using antiserum against catalase and PMP70 (van Grunsven et al., 1999). To examine mitochondria, fibroblasts were incubated with 50nM of MitoTracker Green FM dye (Molecular Probes) and examined with the use of fluorescence microscopy at 488 nm (Waterham et al., 2007). 7 Mutation analysis Mutation analysis was performed by either sequencing all exons plus flanking intronic sequences of the PEX genes amplified by PCR from genomic DNA or by sequencing PEX cdnas prepared from total RNA fractions. Genomic DNA was isolated from skin fibroblasts using the NucleoSpin Tissue genomic DNA purification kit (Macherey-nagel, Germany, Düren). Total RNA was isolated from skin fibroblasts using Trizol (Invitrogen, Carlsbad, CA) extraction, after which cdna was prepared using a first strand cdna synthesis kit for RT-PCR (Roche, Mannheim, Germany). All forward and reverse primers (sequences available on request) were tagged with a -21M13 (5- TGTAAAACGACGGCCAGT-3 ) sequence or M13rev (5 -CAGGAAACAGCTATGACC-3 ) sequence, respectively. PCR fragments were sequenced using -21M13 and M13rev primers by means of BigDye Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA) and analyzed on an Applied Biosystems 377A automated DNA sequencer following the 105

108 Chapter 7 manufacturer s protocol (Applied Biosystems, Foster City, CA, USA). Mutation analysis of PEX1 and PEX3 was performed by cdna sequencing and mutation analysis of PEX2, PEX5L, PEX6, PEX10, PEX11α, PEX11β, PEX11γ, PEX12, PEX13, PEX14, PEX16, PEX19 and PEX26 was performed by genomic DNA sequencing. PEX11β sequence data were compared to the reference PEX11β sequence (NM_003846) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. Genetic complementation The cdnas for PEX11α, PEX11β and PEX11γ were amplified by PCR from a total cdna preparation and subcloned in the eukaryotic pcdna3 expression plasmid (Invitrogen). Constructs were sequenced to exclude PCR-introduced errors. Fibroblasts of the patient were co-transfected with pcdna3 containing either the PEX11α, PEX11β or PEX11γ cdna and a pcdna3 vector expressing egfp- SKL using the AMAXA nucleofector technology (Lonzo). Before transfection, the fibroblasts of the patients were cultured at 37 C. 16 Hours after the transfection, the medium was refreshed and the culture temperature was shifted to 40 C. Cells were examined by means of fluorescence microscopy 4 days after transfection to determine the subcellular localization of the peroxisomal reporter protein egfp- SKL. Two independent transfections per construct were performed and the number of egfp-skl containing peroxisomes was counted in at least 100 cells. 7 Temperature sensitivity of PEX11γ Fibroblasts of the patient and controls were cultured at 37 C and 40 C for 7 days and examined by immunofluorescence microscopy using antibodies against catalase and PMP70 (van Grunsven et al., 1999). After harvesting the cells, total cdna fractions were prepared and real-time RT-PCR performed using the LightCycler 480 SYBR Green I Master kit (Roche) to determine the mrna levels of PEX11γ. Cyclophilin was used as a house-keeping gene to adjust for variations in the amount of input RNA. All samples were analyzed in triplicate. The data were analyzed using linear regression calculations as described previously (Ramakers et al., 2003). In addition, protein homogenates were prepared for western blotting experiments to determine the protein levels of PEX11γ using an antibody against PEX11γ (Proteintech Group). Results Based on clinical presentation, including the occurrence of congenital cataracts, the early sensory neuronal hearing loss and the sensory nerve involvement, the patient was suspected to suffer from a mitochondrial or peroxisomal disorder. In the differential diagnosis, the attacks were considered as a relative sign suggesting a defect in oxidative phosphorylation. However, all mitochondrial parameters were normal in blood. Although the mitochondrial oxidation velocity was slightly reduced in a muscle biopsy of the patient, the ATP and creatine phosphate production from pyruvate were within normal range as were the activities of the mitochondrial complexes in the muscle biopsy. Also all biochemical peroxisomal parameters were mostly normal, including the 106

109 A novel human peroxisome biogenesis disorder affecting peroxisome division Table 1. Biochemical parameters in plasma and erythrocytes. VLCFAs Branched-chain fatty acids Bile acid intermediates Plasmalogens pipecolic acid C26/C22 Phytanic acid Pristanic acid DHCA THCA C29 DMA C16:0 DMA C18:0 (ratio) (μm) (μm) (μm) (μm) (% of total phospholipids) Control Patient Plasmalogens were determined in erythrocytes, all other parameters in plasma. VLCFAs = very long chain fatty acids; DHCA = dihydroxycholestanoic acid; THCA = trihydroxycholestanoic acid; DMA = dimethylacetal Table 2. Biochemical parameters in cultured skin fibroblasts. VLCFAs β-oxidation α-oxidation Enzyme activity C22:0 C24:0 C26:0 C24/C22 C26/C22 C26:0 Pristanic acid Phytanic acid DHAPAT (μmol/g protein) (ratio) (pmol/(hr*mg) protein) (pmol/(hr*mg) protein) (nmol/(2hr*mg) protein) Control range ZS range Patient VLCFAs = very long chain fatty acids; DHAPAT = dihydroxyacetonephosphate acyltransferase 7 107

110 Chapter 7 7 plasma levels of very long chain fatty acids, the branched-chain fatty acids phytanic acid and pristanic acid and the C 27 -bile acid intermediates as were the levels of plasmalogens in erythrocytes (Table 1). Only on one single occasion there was a slightly elevated C26:C22 ratio in plasma of the patient. Studies in cultured skin fibroblasts of the patient also revealed normal levels of very long chain fatty acids, normal β-oxidation rates of cerotic acid (C26:0) and pristanic acid, normal α-oxidation rates of phytanic acid, and normal activity of DHAPAT (Table 2). Interestingly, however, immunofluorescence microscopical analyses of the patient cells using an antibody raised against catalase, a peroxisomal matrix enzyme, and an antibody against PMP70, a peroxisomal membrane protein, revealed a clearly aberrant peroxisomal phenotype. Although in all patient cells peroxisomal membranes were present, in 5-10% of the fibroblasts catalase was not located in peroxisomes but in the cytosol. Furthermore, the peroxisomes varied markedly in size and number. Some fibroblasts showed normal number and normal sized peroxisomes, whereas other fibroblasts showed lower numbers of peroxisomes that were enlarged or elongated compared to control fibroblasts (Figure 1a and b). The enlarged peroxisomes were frequently arranged in rows (Figure 1b), similar as previously observed in DLP1-defective cells (Waterham et al., 2007). Culturing of the patient fibroblasts at 40 C resulted in a complete absence of catalase-containing peroxisomes in approximately 95% of the fibroblasts (Figure 1a), although all cells still contained peroxisomal membrane structures that were enlarged and elongated. The mitochondrial phenotype remained unchanged at 40 C: as in control fibroblasts, the mitochondria were numerous, uniformly sized and randomly dispersed (not shown). a PMP70 Catalase b Control Patient 37 C 40 C Figure 1. Catalase and PMP70 immunofluorescence microscopy in fibroblasts. a, Catalase and PMP70 immunofluorescence microscopy in fibroblasts from the patient cultured at 37 C and 40 C. b, Catalase immunofluorescence microscopy in fibroblasts from a control subject and the patient cultured at 37 C showing the elongated and clustered peroxisomes compared to control fibroblasts. 108

111 A novel human peroxisome biogenesis disorder affecting peroxisome division Figure 2. Sequence and immunoblot analysis of PEX11β. Sequence analysis of the coding region of the PEX11β gene of the patient identified a homozygous mutation, c.64c>t (panel a). The c.64c>t mutation results in the substitution of the glutamine located at position 22 with a nonsense codon. Immunoblot analysis of the peroxisomal membrane protein PEX11β (panel b) in fibroblasts homogenates from control (lane 1) and patient 1 (lane 2). The arrow indicate PEX11β. To exclude that the patient had a defect in any of the 12 PEX genes currently implicated in peroxisome biogenesis disorders, we sequenced all 12 PEX genes, but we did not detect mutations. Because the presence of elongated peroxisomes and enlarged peroxisomes arranged in rows in combination with normal mitochondria morphology pointed to a specific defect in peroxisome division, we considered a defect in one of the 3 different PEX11 genes as a possible cause. We detected no mutations in the PEX11α and PEX11γ but identified a homozygous c.64c>t mutation in the PEX11β gene of the patient (Figure 2). This mutation was detected heterozygous in genomic DNA extracted from blood cells from both parents, confirming the homozygosity in the patient. The c.64c>t mutation changes the glutamine into a nonsense codon (p.q22x). Immunoblot analysis performed with protein homogenates of the patient cells confirmed that this mutation results in a complete absence of the PEX11β protein (Figure 2). 7 PEX11α PEX11β PEX11γ Figure 3. Complementation studies in fibroblasts. Fibroblasts of the patient cultured at 40 C and co-transfected with PEX11α, PEX11β or PEX11γ and the peroxisomal reporter protein GFP-SKL. Overexpression of PEX11α did not complement the peroxisome deficient mutant phenotype. Overexpression of PEX11β reverted the mutant phenotype to wild type phenotype. Overexpression of PEX11γ resulted in partial complementation leading to elongated and enlarged peroxisomes. 109

112 Chapter 7 Table 3. Number of cells with catalase import competent peroxisomes of patient fibroblasts cultured at 40 C and transfected with either an empty vector or a vector expressing PEX11α, PEX11β or PEX11γ. Number of cells Catalase positive peroxisomes Catalase negative peroxisomes pcdna pcdna3-pex11α pcdna3-pex11β pcdna3-pex11γ Overexpression of wild type PEX11β in fibroblasts of the patient cultured at 40 C reverted the mutant peroxisome-deficient phenotype to the normal wild-type phenotype (Figure 3). Overexpression of PEX11α in fibroblasts of the patient cultured at 40 C did not complement the peroxisome-deficient mutant phenotype at 40 C (Figure 3). Remarkably, however, overexpression of PEX11γ in fibroblasts of the patient cultured at 40 C showed partial complementation and resulted in elongated and enlarged peroxisomes in approximately 50% of the transfected cells (Figure 3 and Table 3). This latter phenotype looked very similar to the phenotype observed in the patient fibroblasts cultured at 37 C, suggesting that the presence of the enlarged, elongated catalase-containing peroxisomes is dependent on the levels of PEX11γ. This is in line with the observation that both the mrna and protein levels of PEX11γ are markedly decreased in cells cultured at 40 C when compared to cells cultured at 37 C (Figure 4). a Control Patient b PEX11γ Actin Figure 4. mrna and protein levels of PEX11γ. mrna and protein levels of PEX11γ in control and patient fibroblasts cultured at 37 C and 40 C for 7 days. a, After 7 days the cells are harvested for RNA isolation followed by cdna synthesis and real-time RT-PCR. The PEX11γ mrna levels are presented as the ratio of PEX11γ/cyclofilin, the control ratios are the average of 3 different control cell lines. b, Immunoblot analysis of PEX11γ in homogenates from control cells cultured at 37 C (lane 1), control cells cultured at 40 C (lane 2), patient cells cultured at 37 C (lane 3) and patient cells cultured at 40 C (lane 4). 110