Sanne Merel

Petra Ebberink

Jeannette Mooyer

Janet Gootjes

Ronald J A Koster

Hans Wanders

Waterham

Merel S Ebberink

Petra A W Mooijer

Jeannette Gootjes

Janet Koster

Ronald J A Wanders

PhD Hans R Waterham
email: h.r.waterham@amc.uva.nl

Genetic

Keywords: Zellweger Syndrome, PEX genes, PEX genes, peroxisome, biogenesis Zellweger Syndrome, PEX genes, genetic complementation, peroxisome, biogenesis

Introduction

Peroxisome biogenesis disorders (MIM 601539) include the Zellweger syndrome spectrum (ZSS) disorders and Rhizomelic Chondrodysplasia Punctata type I [START_REF] Weller | Peroxisome biogenesis disorders[END_REF]. 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 [START_REF] Braverman | Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype[END_REF][START_REF] Motley | Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1[END_REF]. The ZSS disorders include the Zellweger syndrome (ZS, MIM 214100), neonatal adrenoleukodystrophy (NALD, MIM 202370) and infantile Refsum disease (IRD, MIM 266510), 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 [START_REF] Steinberg | Peroxisome biogenesis disorders[END_REF]. 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 [START_REF] Wanders | Biochemistry of mammalian peroxisomes revisited[END_REF][START_REF] Weller | Peroxisome biogenesis disorders[END_REF]).

Many human PEX genes have been identified on the basis of sequence similarity with yeast PEX genes identified by functional complementation of peroxisome-deficient yeast mutants. At present more than 30 different peroxins have been identified in different yeast species, including Saccharomyces cerevisiae and Yarrowia lipolytica. The peroxins are involved in different peroxisomal processes, such as the formation of peroxisomal membranes, peroxisomal growth, fission and proliferation, and import of matrix proteins [START_REF] Platta | The peroxisomal protein import machinery[END_REF]. Of these yeast peroxins, 19 have been implicated in peroxisome protein import and biogenesis, whereas in humans, so far only 13 peroxins have been 4 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 a majority of cell lines. We here report the results of our genetic complementation studies in more than 600 cell lines from patients 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.

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 VLCFA analysis, C26:0 and pristanic acid β-oxidation, phytanic acid α-oxidation and dihydroxyacetonephosphate acyltransferase (DHAPAT) activity analysis, and the absence of peroxisomes assessed by on catalase immunofluorescence (IF) microscopy [START_REF] Wanders | Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders[END_REF]. The cell lines 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. 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.

Genetic complementation assays

To assign cell lines to genetic complementation groups, we used a previously described polyethylene glycol (PEG)-mediated cell fusion assay [START_REF] Brul | Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis[END_REF] and a PEX cDNA transfection assay, specifically developed for this purpose. In the latter assay, ZSS cell lines are co-transfected separately with one of the 12 different PEX cDNAs subcloned in the pcDNA3 expression vector (Invitrogen, Carlsbad, CA) and an eGFP-SKL vector [START_REF] Waterham | A lethal defect of mitochondrial and peroxisomal fission[END_REF]. The eGFP-SKL vector expresses eGFP tagged with the peroxisomal targeting signal -SKL, which allows immediate visual detection of peroxisomes when present. The 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 performed the transfections of cell lines in three subsequent series based on the estimated frequency of the different PEX gene defects. In the first series, we transfected cell lines separately with PEX1, PEX6 and PEX12. Cell lines that were not functionally complemented were then transfected separately with PEX2, PEX10 and PEX26 and finally with PEX5, PEX13 and PEX14. Transfection with PEX3, PEX16 and PEX19 was performed when the patient fibroblasts showed no peroxisomal membrane remnants based on immunofluorescence microscopy [START_REF] Van Grunsven | Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis[END_REF] with antibodies against ALDP (Euromedex, France, Souffelweyersheim), 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 (see also figure 1).

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 (Supp tables S1-11) 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 Mutation analysis of PEX1 (GenBank accession number NM_000466.2) was performed by sequencing the coding region of the PEX1 cDNA and/ or exons amplified from gDNA or by RFLP analysis to identify the frequently occurring c.2528G>A mutation. The RFLP analysis involves PCR amplification using a specific primer set (see Supp Table S1) of a 130-bp genomic DNA fragment encompassing the c.2528G/A nuctleotide, followed by restriction analysis with ClaI. The c.2528G>A mutation creates a restriction recognition site for ClaI and thus can be screened for by RFLP analysis. Mutations in the PEX2 gene were identified by sequencing the coding exon 4 from gDNA (NM_000318.2). The PEX3 (NM_003630.2) gene was analyzed by sequencing of the coding region of the PEX3 cDNA. Mutation analysis of

PEX5L (X84899), PEX10 (NM_002617.3), PEX12 (NM_000286.2), PEX13 (NM_002618.2), PEX14 (NM_004565.2), PEX16 (NM_004813.2), PEX19 (NM_002857.

Results and Discussion

Genetic complementation

Initially, we used a previously reported cell fusion complementation assay [START_REF] Brul | Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis[END_REF].

Because this assay is rather laborious, we developed a more rapid and 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., 2004b). 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 from Western European origin and thus the spectrum of PEX gene defects primarily relates to the Western European population.

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 [START_REF] Krause | Rational diagnostic strategy for Zellweger syndrome spectrum patients[END_REF]. This suggests that, in contrast to yeast where 19 different PEX genes have been implicated, peroxisome 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 gene [START_REF] Waterham | A lethal defect of mitochondrial and peroxisomal fission[END_REF]. In addition, defects in certain PEX genes may also be without consequences due to gene redundancy. For example, recently 3 human proteins have been reported with a similar function as the yeast PEX4 gene, which is associated with complete peroxisome deficiency in yeast [START_REF] Grou | Members of the E2D (UbcH5) Family Mediate the Ubiquitination of the Conserved Cysteine of Pex5p, the Peroxisomal Import Receptor[END_REF]. Considering that these proteins 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 [START_REF] Grou | Members of the E2D (UbcH5) Family Mediate the Ubiquitination of the Conserved Cysteine of Pex5p, the Peroxisomal Import Receptor[END_REF]. Finally, although we consider this unlikely, we cannot exclude the possibility that a defect in certain unannotated PEX genes may be embryonic lethal and thus will not be diagnosed.

Mutation analysis

After the assignment of patient cell lines to genetic complementation groups, we performed mutation analysis of the respective PEX genes in 393 of the cell lines. For most genes all coding exons plus flanking intron sequences were analysed by sequencing. In principle, this method will identify all missense and nonsense mutations, small deletions, small duplications and small insertions located in the analysed fragments. However, the presence of large, heterozygous deletions or duplications, or pathogenic mutations located outside the analysed fragments cannot be excluded. 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 a given PEX gene that was demonstrated to be defective by the fact that peroxisome biogenesis in the corresponding cell line is restored after transfection with the respective PEX cDNA (i.e. a functional assay). 2) The sequence variants are not listed in the SNP database or encountered in more than 100 control chromosomes. 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 [START_REF] Maxwell | Novel PEX1 coding mutations and 5' UTR regulatory polymorphisms[END_REF]. 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;[START_REF] Maxwell | Novel PEX1 coding mutations and 5' UTR regulatory polymorphisms[END_REF]. Patients homozygous for the G843D mutation usually present with the milder IRD phenotype. Cells of these patients often display peroxisomal mosaicism at 37°C, but a complete peroxisome deficiency when cultured at 40°C. The second most common mutation in PEX1 is c.2079_2098insT, which results in a frame shift predicted to cause a truncated PEX1 protein [START_REF] Collins | Identification of a common PEX1 mutation in Zellweger syndrome[END_REF], although Northern blot analysis showed that this mutation results in low steady state PEX1 mRNA levels [START_REF] Maxwell | Novel PEX1 coding mutations and 5' UTR regulatory polymorphisms[END_REF]. Patients homozygous for this mutation are affected with the severe ZS phenotype. The c.2926delA 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 88 different mutations have now been identified in the PEX1 gene, of which 34 have not been reported previously. In two cell lines we found three heterozygous mutations after sequencing gDNA (Table 1). In the first cell line the following 3 heterozygous mutations were found, p.T526A, p.911_912delCT and p.1885_1886delGT, of which the last two have been reported as pathogenic in literature. The consequence of the p.T526A mutation is unclear, but the threonine at position is a conserved amino acid in Pan troglodytes Deleted: 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 of mRNA stability. ¶ program Polyphen predicts this amino acid substitution as possibly damaging. In the second cell line, the heterozygous p.V336A, p.S555P and p.R949W missense mutations all affect well conserved amino acids and, according to Polyphen, are predicted to be benign, possibly damaging and probably damaging, respectively, suggesting that p.V336A may be a rare sequence variation. All the novel identified missense mutations are not listed in the SNP database, have not been detected in more than 100 control chromosomes and in fact all affect (well) conserved amino acids. In addition, Polyphen predicts these amino acid substitutions as either possibly damaging or probably damaging.

The PEX2 (MIM170993) gene was the first gene reported to be mutated in ZSS patients. PEX2 is an integral membrane protein with two transmembrane domains and a zincbinding motif, probably involved in the poly-ubiquitination of the peroxisomal import receptor PEX5 [START_REF] Platta | Pex2 and pex12 function as protein-ubiquitin ligases in peroxisomal protein import[END_REF]. The PEX2 gene contains four exons, but the entire coding region 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 [START_REF] Gootjes | Resolution of the molecular defect in a patient with peroxisomal mosaicism in the liver[END_REF]Gootjes et al., 2004a). In 3 different cell lines, sequence analysis of exon 4 identified only one heterozygous mutation (table 1). We did not analyze the remaining exons, promoter sequence or PEX2 cDNA of these cell lines. In this study, six novel mutations have been identified, making a total of 17 different mutations currently identified in the PEX2 gene (table 3). Two novel missense mutation are identified which are not listed in the SNP database, and both affect (well) conserved amino acids. In addition, Polyphen predicts these amino acid substitutions as probably damaging. MIM603164) is an integral membrane protein with two putative membranespanning domains. The exact function of PEX3 is unclear, but it has been 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 [START_REF] Ghaedi | PEX3 is the causal gene responsible for peroxisome membrane assembly-defective Zellweger syndrome of complementation group G[END_REF][START_REF] Muntau | Defective peroxisome membrane synthesis due to mutations in human PEX3 causes Zellweger syndrome, complementation group G[END_REF]. In total, six different mutations in PEX3 were identified of which four have been reported previously (table 4).

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), [START_REF] Saidowsky | The di-aromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14[END_REF][START_REF] Weller | Peroxisome biogenesis disorders[END_REF], 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 [START_REF] Dodt | Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p[END_REF]. 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. We recently published and discussed the mutations we identified in the PEX5 gene elsewhere (Ebberink et al., 2009b). These mutations combined with previously reported mutations are listed in table 5.

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., 2009a). PEX10 (MIM602859) is an integral membrane protein and contains two transmembrane domains and a C-terminal zinc-binding motif, similar as 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 [START_REF] Warren | Phenotype-genotype relationships in PEX10deficient peroxisome biogenesis disorder patients[END_REF]. Combined with previously reported PEX10 mutations, 25 different mutations have been identified in the PEX10 gene of which nine have not been reported previously (table 6). The six novel missense mutations all affect well conserved amino acids and Polyphen predicts these amino acid substitutions as probably damaging. Sequence analysis of the exons plus flanking intronic sequences of the PEX10 gene identified in one patient only one heterozygous mutation. We did not analyze the PEX10 cDNA in this cell line.

Mutations in the PEX12 gene are the third common cause of a ZSS disorder: 9% of our patients group has mutations in PEX12. PEX12 is one of the RING (abbreviation for really interesting new gene) finger proteins; it contains two transmembrane domains and a Cterminal 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 for this mutation present with an atypical biochemical phenotype in skin fibroblasts: the peroxisomal parameters are mostly normal to slightly abnormal. The mutation causes a temperaturesensitive 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., 2004c). We identified two novel missense mutations both affecting well conserved amino acids and Polyphen predicts these amino acid substitutions as probably damaging.

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 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 [START_REF] Liu | PEX13 is mutated in complementation group 13 of the peroxisome-biogenesis disorders[END_REF]Shimozawa et al., 1999a). One novel missense mutation was identified which affects a well conserved amino acid and is probably damaging according to Polyphen.

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 aminoterminal WxxxF/Y motifs of PEX5. More recently, it was proposed that PEX14 is also the site from which PEX5 leaves the peroxisomal compartment [START_REF] Azevedo | Pex14p, more than just a docking protein[END_REF]. 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 which the fibroblasts displayed a defect in import of peroxisomal matrix and membrane proteins, resulting in a total absence of peroxisomal remnants [START_REF] Honsho | Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D[END_REF][START_REF] Shimozawa | A novel aberrant splicing mutation of the PEX16 gene in two patients with Zellweger syndrome[END_REF], and very recently, several patients with a relatively mild clinical phenotype of which the fibroblasts showed enlarged, import-competent peroxisomes [START_REF] Ebberink | Identification of an unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene[END_REF]. In total eight different mutations have been identified in the PEX16 gene of which one has not been reported previously (table 10).

The PEX19 (MIM600279) gene encodes a mainly cytosolic protein that binds many peroxisomal membrane proteins, suggesting that PEX19 functions as a peroxisomal receptor for peroxisomal membrane proteins [START_REF] Sacksteder | PEX19 binds multiple peroxisomal membrane proteins, is predominantly cytoplasmic, and is required for peroxisome membrane synthesis[END_REF]. The patients with a defect in PEX19 presented with a severe ZS phenotype and the fibroblasts displayed the absence of peroxisomal ghosts [START_REF] Matsuzono | Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly[END_REF]. 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., 2003b;Matsumoto et al., 2003a). 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).

Conclusions

In summary, we developed an efficient genetic complementation assay, which allows rapid identification of the defective PEX gene in fibroblast cell lines of ZSS patients. This assay has been incuded 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. These mutations appear homozygous, but a genomic deletion of one allele cannot be excluded.

2

This number of cell lines includes 2 cell lines with 3 heterozygous mutations.

3 Mutation analysis of the PEX1 gene by RFLP analysis identified the p.G843D mutation heterozygous in 38 patients; the PEX1 gene in these patients was not further screened by sequence analysis. 4 Only coding exon 4 was sequenced. 5 We did not analyze the corresponding cDNA in these cell lines [START_REF] Gartner | Mutations in PEX1 in peroxisome biogenesis disorders: G843D and a mild clinical phenotype[END_REF][START_REF] Portsteffen | Human PEX1 is mutated in complementation group 1 of the peroxisome biogenesis disorders[END_REF][START_REF] Preuss | PEX1 mutations in complementation group 1 of Zellweger spectrum patients correlate with severity of disease[END_REF][START_REF] Reuber | Mutations in PEX1 are the most common cause of peroxisome biogenesis disorders[END_REF] p.V659D 1 this study 12 c.1991T>C p.L664P [START_REF] Tamura | Phenotype-genotype relationships in peroxisome biogenesis disorders of PEX1defective complementation group 1 are defined by Pex1p-Pex6p interaction[END_REF] 12 c.2008C>A p.L670M 1 [START_REF] Walter | Disorders of peroxisome biogenesis due to mutations in PEX1: phenotypes and PEX1 protein levels[END_REF] 12 c.2034_2035delCA p.H678QfsX15 1 [START_REF] Steinberg | The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum[END_REF]) 12 c.2071+1G>T -5 [START_REF] Walter | Disorders of peroxisome biogenesis due to mutations in PEX1: phenotypes and PEX1 protein levels[END_REF] 13 c.2083_2085delATG p.M695del [START_REF] Rosewich | Genetic and clinical aspects of Zellweger spectrum patients with PEX1 mutations[END_REF]) 13 c.2085-2089delGATAA p.M695IfsX45 [START_REF] Steinberg | The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum[END_REF] 13 c.2097_2098insT p.I700fsX41 16 32 [START_REF] Collins | Identification of a common PEX1 mutation in Zellweger syndrome[END_REF][START_REF] Maxwell | Novel PEX1 coding mutations and 5' UTR regulatory polymorphisms[END_REF][START_REF] Preuss | PEX1 mutations in complementation group 1 of Zellweger spectrum patients correlate with severity of disease[END_REF][START_REF] Steinberg | The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum[END_REF] [START_REF] Collins | Identification of a common PEX1 mutation in Zellweger syndrome[END_REF][START_REF] Maxwell | Novel PEX1 mutations and genotype-phenotype correlations in Australasian peroxisome biogenesis disorder patients[END_REF][START_REF] Preuss | PEX1 mutations in complementation group 1 of Zellweger spectrum patients correlate with severity of disease[END_REF][START_REF] Steinberg | The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum[END_REF][START_REF] Walter | Disorders of peroxisome biogenesis due to mutations in PEX1: phenotypes and PEX1 protein levels[END_REF] Imamura et al., 1998b;[START_REF] Shimozawa | Standardization of complementation grouping of peroxisome-deficient disorders and the second Zellweger patient with peroxisomal assembly factor-1 (PAF-1) defect[END_REF][START_REF] Steinberg | The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum[END_REF] 4 c.373C>T p.R125X (Imamura et al., 1998b;[START_REF] Shimozawa | A novel mutation, R125X in peroxisome assembly factor-1 responsible for Zellweger syndrome[END_REF][START_REF] Steinberg | The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum[END_REF] [START_REF] Muntau | Defective peroxisome membrane synthesis due to mutations in human PEX3 causes Zellweger syndrome, complementation group G[END_REF]Shimozawa et al., 2000a;[START_REF] South | Inhibitors of COPI and COPII do not block PEX3-mediated peroxisome synthesis[END_REF] 10 c.856c>t p.R286X 1 [START_REF] Dursun | Zellweger syndrome with unusual findings: non-immune hydrops fetalis, dermal erythropoiesis and hypoplastic toe nails[END_REF] 11 c.942-8T>G p.S314RfsX3 [START_REF] Ghaedi | PEX3 is the causal gene responsible for peroxisome membrane assembly-defective Zellweger syndrome of complementation group G[END_REF][START_REF] Muntau | Defective peroxisome membrane synthesis due to mutations in human PEX3 causes Zellweger syndrome, complementation group G[END_REF] Reference sequence of PEX3: GenBank accession number NM_003630. Ebberink et al., 2009b;Shimozawa et al., 1999b S1. Primer sets used for PEX1 mutation analysis.

  with the NHDF Nucleofector Kit (Amaxa, Cologne, Germany)

  by means of BigDye Terminator v1.1 Deleted: used the following transfection order of PEX expression vectors based on the estimated frequency of the different PEX gene defects: 1) (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).

  2) and PEX26 (NM_017929.4) were performed by sequencing of all exons plus flanking intronic sequences amplified from gDNA. Sequences were compared with the indicated reference sequences with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. The possible consequences of missense mutations were analyzed using the Polyphen prediction software (http://genetics.bwh.harvard.edu/pph/)

  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

  , Canis familiaris (dog) and Gallus gallus domesticus (chicken) and the software

  N-terminal part of yeast PEX13 interacts with PEX7. Table

Figure 1 .

 1 Figure legend 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).

  2. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

  ) Reference sequence of PEX5L: GenBank accession number X84899. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

  2. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

Table 1 .

 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 Frequency		Number of cell lines sequenced and mutations
		(Number of cell lines)	(%)	Total Homozygous 1 2x Heterozygous 1x Heterozygous
	PEX1	358	58	180	74	68 2	38 3
	PEX2	23	4	22	17	2	3 4,5
	PEX3	3	<1	3	3
	PEX5	13	2	13	13
	PEX6	97	16	77	45	32
	PEX10	19	3	18	10	7	1 5
	PEX12	54	9	43	29	14
	PEX13	10	1	7	7
	PEX14	3	<1	2	1		1 5
	PEX16	8	1	8	8
	PEX19	4	<1	3	3
	PEX26	21	3	17	15	2
	Total	613		393
	1

Table 2 .

 2 Mutations in the PEX1 gene.

	Mutations

  ; Walter

	Page 27 of 48		Human Mutation
				et al., 2001)
	13 c.2224C>T	p.Q742X	2	this study
	13 c.2227_2416del	p.E743NfsX2fs		(Preuss et al., 2002)
	14 c.2364G>A	Deletion exon 14	1	this study
	14 c.2368C>T	p.R790X	1	(Maxwell et al., 2002)
	14 c.2383C>T	p.R795X	1	(Collins and Gould, 1999)
	14 c.2387T>C 14 c.2391_2392delTC 14 c.2392C>G 15 c.2528G>A	p.L796P p.R798SfsX35 p.R798G F o r P e e r R e v i e w 1 (Rosewich et al., 2005) (Maxwell et al., 2005) 1 (Maxwell et al., 2002) p.G843D 42 62
				Deleted: 13	... [2]
				Deleted: 5
				Deleted: 3
			John Wiley & Sons, Inc.

  GenBank accession number NM_000466.2. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG. 1 Possibly this mutation is a rare sequence variation, for details see text.

	c.2927-15T_3208-
	342AdelinsATAGTATAGA &	del ex19/20		2	this study
	c.3849+779_3849+2910
	19 c.2966T>C	p.I989T			(Maxwell et al., 2005)
	19 c.2992C>T	p.R998X			(Maxwell et al., 2002)	Deleted: p.V788V
	19 c.2993G>A	p.R998Q			(Maxwell et al., 2005)	Deleted: 2
	19 c.3022_3024delCCT	p.P1008del			(Steinberg et al., 2004)
	20 c.3038G>A	p.R1013H			(Rosewich et al., 2005)
	20 c.3180_3181insT	p.G1061WfsX16			(Collins and Gould, 1999)
	20 c.3207+1G>C	-			(Collins and Gould, 1999)
	21 c.3287C>G	p.S1096X			(Rosewich et al., 2005)	Deleted: 4
	15 c.2537_2545delATGAAGTTAinsTCATGGT p.H846_R849delinsLfsX53 21 c.3378C>G p.Y1126X 23 c.3692-3695delAGTC p.Q1231HfsX3		1	(Steinberg et al., 2004) (Preuss et al., 2002) (Yik et al., 2009; Rosewich et al., 2005)	Deleted: 9
	15 14bpinsex15 24 c.3850T>C	-p.X1284QnextX29		1	(Reuber et al., 1997) (Walter et al., 2001)
	16 c.2614C>T Reference sequence of PEX1:	p.R872X	2	1	(Preuss et al., 2002)	Deleted: 1
	16 c.2633_2635 delTGT	p.L879del	1	1	this study
	16 c.2636T>C	p.L879S		3	this study	Deleted: 2
	16 c.2654C>G	p.T885R		1	this study
	17 c.2730delA	p.l910fsX50	1	4	(Walter et al., 2001)
	17 c.2760delA	p.A921LfsX40	1		this study
	18 c.2814_2818delCTTTG	p.F938LfsX2			(Preuss et al., 2002)
	18 c.2845C>T	p.R949W		1	this study
	18 c.2846G>A	p.R949Q		2	(Walter et al., 2001)
	18 c.2916delA	p.G973AfsX16	1	4	(Maxwell et al., 2002; Preuss et al., 2002; Steinberg et al., 2004)
	18 c.2926+1G>A	-		1	(Steinberg et al., 2004)
	18 c.2926+2_2926+3insT	-			(Collins and Gould, 1999)
	18 c.2926+2T>C	-			(Walter et al., 2001)

Table 3 .

 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			(Krause et al., 2006)
	4	c.163 G>A	p.E55K			(Imamura et al., 1998b; Steinberg et al., 2004)
	4	c.273delT	p.N92TfsX2			(Steinberg et al., 2004)
	4	c.279_283delGAGAT	p.R94SfsX5	2	1	(Gootjes et al., 2004a)
	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	(

  GenBank accession number NM_000318.2. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

					)
	4	c.550delC	p.R184VfsX8	3	(Shimozawa et al., 2000b)
	4	c.642delG	p.K215SfsX2	1	(Shimozawa et al., 2000b)
	4	c.669G>A	p.W223X	2	(Gootjes et al., 2004a)
	4	c.739T>C	p.C247R	1	(Gootjes et al., 2004a)
	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	(Krause et al., 2006)
	4	c.866insA	p.S289fsX36	1	this study
	Reference sequence of PEX2:

Table 4 .

 4 Mutations in the PEX3 gene.

		Mutations	Number of patients identified in this study
	Exon Nucleotide	Amino acid	Homozygous	Heterozygous	Reference
	2	c.157C>T	p.R53X			(South et al., 2000)
	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			(

Table 5 .

 5 Mutations in the PEX5 gene.

				Number of patients identified in
		Mutations	this study
	Exon Nucleotide	Amino acid	Homozygous	Heterozygous Reference
	5	c.548_552-55delins238inv	p.D150GfsX33	1	(Ebberink et al., 2009b)
	7	c.826C>T	p.R276X	1	(Ebberink et al., 2009b)
	10	c.1090C>T	p.Q364X	1	(Ebberink et al., 2009b)
		[c.1244A>G]+[c.548-549dupATCG;
	11	c.604G>C]	p.N415S	1	(Ebberink et al., 2009b)
	i12	c.1394+5G>A	p.R394S, r.395_465del	1	(Ebberink et al., 2009b)
	12	c.1258C>T	p.R420X	2	(Ebberink et al., 2009b)
	12	c.1279C>T	p.R427X		(Dodt et al., 1995)
	i14	c.1561-2A>C	r.1561_1718del and r.1561_1584del	1	(Ebberink et al., 2009b)
	14	c.1578T>G	pN526K	2	(Dodt et al., 1995; Ebberink et al., 2009b)
	14	c.1669C>T	p.R557W	1	(Ebberink et al., 2009b)
	15	c.1799C>G	p.S600W	1	(

Table 6 .

 6 Mutations in the PEX10 gene. GenBank accession number NM_002617.3. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

	Page 35 of 48				Human Mutation
		Mutations	Number of patients identified in this study
	Exon	Nucleotide	Amino acid	Homozygous	Heterozygous	Reference
	1	c.2T>C	p.M1T		1	(Regal et al., 2010)
	1	c.3G>A	p.M1I		1	this study
	1	c.4delG	p.A2PfsX9			(Steinberg et al., 2004)
	1	c.13_28del, ins20bp	p.A5PfsX46			(Warren et al., 2000)
	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			(Steinberg et al., 2009)
	3	c.352C>T	p.Q118X	1		this study
	3	c.373C>T	p.R125X			(Warren et al., 1998)
	i3	c.600+1G>A	-	1		(Warren et al., 1998)
	4	c.646_647insA	p.S216KfsX123	1		this study
	4	c.697delG	p.A213PfsX131	1		this study
	4	c.704_705insA	p.L236AfsX102	2	2	(Steinberg et al., 2004; Warren et al., 2000)	Deleted: 1
	4	c.730C>T	p.R244X		1	(Krause et al., 2006; Steinberg et al., 2004)
	5	c.814_815delCT	p.L272VfsX65	1	1	(Okumoto et al., 1998a)
	5	c.830T>C	p.L277P			(Steinberg et al., 2009)
	5	c.835G>T	p.E279X		1	(Steinberg et al., 2004)
	5	c.868C>G	p.H290D			(Steinberg et al., 2004)
	5	c.870C>G	p.H290Q			(Warren et al., 1998)
	5	c.881G>A	p.W294X			(Krause et al., 2006)
	5	c.892G>T	p.C301F			(Steinberg et al., 2004)
	6	c.919T>C	p.C307R	1		this study
					John Wiley & Sons, Inc.

36 of 48 John Wiley & Sons, Inc. Human Mutation F o r P e e r R e v i e wTable 7 .

 7 Mutations in the PEX12 gene. GenBank accession number NM_000286.2. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

	Page 37 of 48					Human Mutation
	3	c.744_745insT		p.T249YfsX14		1	(Chang and Gould, 1998)
	3 Exon Nucleotide c.775C>T 3 c.875_876delCT	Mutations	p.Q259X Amino acid p.S292X	Number of patients identified in 1 this study Homozygous Heterozygous 1	this study (Okumoto et al., 1998b) Reference (Chang et al., 1999; Gootjes et al., 2004c; Steinberg et al.,
	1 3	c.16dupG c.887_888delTC		p.A6GfsX11 p.L296fs3007X	1	1 1	this study 2004)
	1 3	c.26_27delCA c.887delT		p.T9SfsX7 p.L296PfsX5			(Chang and Gould, 1998) (Steinberg et al., 2004)
	1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 Reference sequence of PEX12: c.102A>T c.126+1G>T c.202_204delCTT c.220T>G c.260_261insAA c.268_271delAAGA c.273A>T c.308_309insT c.378_380delTTC c.445_454delTCTTCCCGCT c.460C>T c.478_479delCA c.531_533delACA c.533_535delAAC c.538C>T c.541_542insT c.604C>T c.625C>T c.888_889delCT c.920G>A c.949C>T c.959C>T c.1047_1049delACA	p.R34S -p.L68del p.Y74D p.Y87X p.K90EfsX3 p.R91S p.L103FfsX3 p.L126del p.S149GfsX15 p.R154X p.Y156X p.Q178del p.Q178del p.R180X p.Y181LfsX37 p.R202X p.Q209X p.L297TfsX12 p.C307Y p.L317F p.S320F p.Q349del F o r P e e r R e v i e w 1 (Zeharia et al., 2007) 3 5 (Chang and Gould, 1998) (Chang and Gould, 1998) 1 this study (Steinberg et al., 2004) (Chang and Gould, 1998) 1 (Gootjes et al., 2004c) 1 (Gootjes et al., 2004c) 1 this study 1 this study 1 this study 1 this study (Yik et al., 2009) 1 1 (Steinberg et al., 2004) 2 (Chang et al., 1999; Okumoto et al., 1998b) (Steinberg et al., 2004) 3 (Gootjes et al., 2004c) 2 (Gootjes et al., 2004c) 1 3 (Chang and Gould, 1998; Steinberg et al., 2004) 1 this study 1 (Gootjes et al., 2004d) 13 2 (Steinberg et al., 2004) 2 (Steinberg et al., 2004)	Deleted: 2 Deleted: 2
	i2	c.681-2A>C		-			(Steinberg et al., 2004)
	3	c.684_687delTAGT	p.S222RfsX3			(Chang and Gould, 1998)
	3	c.691A>T		p.K231X			(Okumoto and Fujiki, 1997)
	3	c.733_734dupGCCT	p.L245CfsX19		1	(Chang et al., 1999; Steinberg et al., 2004)
						John Wiley & Sons, Inc.

Table 8 .

 8 Mutations in the PEX13 gene. GenBank accession number NM_0002618.2. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG. 1 Deletion of 147,308 bp that included the PEX13 gene and 70,094 bp upstream and 45,692 bp downstream of the PEX13 the 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			(Al-Dirbashi et al., 2009)
	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		(Shimozawa et al., 1999a)
	4	c.937T>G	p.W313G			(Krause et al., 2006)
	4	c.970G>T	p.G324X			(Shimozawa et al., 1999a)
	4	c.977T>C	p.I326T	1		(Liu et al., 1999; Shimozawa et al., 1999a)
	4	c.980c>g	p.P327R	1		this study
	1-4	c.1_1212 1	-			(Al-Dirbashi et al., 2009)
	Reference sequence of PEX13:

Table 9 .

 9 Mutations in the PEX14 gene. GenBank accession number NM_004565.2. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

		Mutations	Number of patients identified in this study
	Exon Nucleotide	Amino acid	Homozygous	Heterozygous	Reference
	3 c.85-?_170+?del	p.[Ile29_Lys56del;Gly57GlyfsX2]	1		(Huybrechts et al., 2008)
	7 c.553C>T	p.Gln185Ter			(Shimozawa et al., 2004)
	7 c.585+1G>T	r.585+1_37ins		1	this study
	Reference sequence of PEX14:

Table 10 .

 10 Mutations in the PEX16 gene. GenBank accession number NM_0004813.

		Mutations		Number of patients identified in this study
	Exon	Nucleotide	Amino acid	Homozygous	Heterozygous	Reference
	i5	c.460+2 insG	-	1		this study
	6	c.526C>T	p.R176X			(Honsho et al., 1998; South and Gould, 1999)
	8	c.753_755delTGT	p.V252del	1		Ebberink 2010
	9	c.865C>A	p.P289T	1		Ebberink 2010
	i11	c.952+118_1011+80		1		Ebberink 2010
	i10	c.952+2T>C	R298fsX38	2		(Shimozawa et al., 2002)
	11	c.984delG	p.I330SfsX27	1		Ebberink 2010
	11	c.992A>G	p.Y331C	1		Ebberink 2010
	Reference sequence of PEX16:

Table 11 .

 11 Mutations in the PEX19 gene. GenBank accession number NM_0002857.2. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

		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	(Matsuzono et al., 1999)
	Reference sequence of PEX19:

Table 12 .

 12 Mutations in the PEX26 gene. GenBank accession number NM_017929.4. Nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

				Number of patients identified in
		Mutations	this study
	Exon Nucleotide	Amino acid	Homozygous	Heterozygous	Reference
	2	c.2T>C	p.M1T			(Matsumoto et al., 2003b; Weller et al., 2005)
	2	c.34_35insC	p.L12PfsX103	2		(Matsumoto et al., 2003b)
	2	c.37_38delAG	p.R13GfsX164			(Steinberg et al., 2004)
	2	c.73_79delGTGCGCG	p.V25RfsX55	1		this study
	2	c.131T>C	p.L44P		1	(Weller et al., 2005)
	2	c.134T>C	p.L45P			(Matsumoto et al., 2003b; Weller et al., 2005)
	2	c.185G>A	p.W62X	1		this study
	2	c.192_216del	p.S64RfsX10			(Steinberg et al., 2004)
	i2	c.230+1G>T	p.T77fsX139			(Weller et al., 2005)
	3	c.254_255insT	p.L85LfsX93			(Matsumoto et al., 2003b; Weller et al., 2005)
	3	c.265G>A	p.G89R			(Matsumoto et al., 2003b)
	3	c.292C>T	p.R98W	10	2	(Matsumoto et al., 2003b; Steinberg et al., 2004; Weller et al., 2005)
	3	c.296G>A	p.W99X			(Steinberg et al., 2004)
	3	c.315g>a	p.W105X	1		this study
	3	c.350C>T	p.P117L			(Weller et al., 2005)
	3	c.353C>G	p.P118R			(Steinberg et al., 2004)
	i3	c.371+2T>C	-			(Steinberg et al., 2004)
	4	c.426_548dup122bpinsT	p.A143_V182dup+G183V			(Weller et al., 2005)
	4	c.574C>T	p.R192X		1	(Steinberg et al., 2004)
	i4	c.667+2T>C	-			(Steinberg et al., 2004)
	5	r.668_814	p.G223_P271del			(Matsumoto et al., 2003b)
	6	c.862delC	p.R288AfsX366			(Weller et al., 2005)
	Reference sequence of PEX26:

John Wiley & Sons, Inc.Human Mutation

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

Amplicon Sequence (5'→ → → →3')

1 Exons Regions of cDNA

All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S2. Primer sets used for PEX2 mutation analysis.

Supplemental Table

All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S3. Primer sets used for PEX3 mutation analysis.

Supplemental Table

Amplicon Sequence (5'→3') 1

Regions of cDNA

All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S4. Primer sets used for PEX5 mutation analysis Amplicon Sequence (5'→ → → →3')

Supplemental Table

All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S5. Primer sets used for PEX10 mutation analysis.

Supplemental Table

Amplicon Sequence (5'→ → → →3')

All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S6. Primer sets used for PEX12 mutation analysis.

Supplemental Table

Amplicon Sequence (5'→ → → →3')

All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S7. Primer sets used for PEX13 mutation analysis

Supplemental Table

Amplicon

Sequence (5'→ → → →3') All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S8. Primer sets used for PEX14 mutation analysis Amplicon Sequence (5'→ → → →3')

Supplemental Table

All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S9. Primer sets used for PEX16 mutation analysis. All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S10. Primer sets used for PEX19 mutation analysis Amplicon Sequence (5'→ → → →3')

Supplemental Table

Supplemental Table

All forward and reverse primers were tagged with a -21M13 (5'-GTAAAACGACGGCCAGT-3') sequence or M13rev (5'-CAGGAAACAGCTATGACC-3') sequence. S11. Primer sets used for PEX26 mutation analysis.

Supplemental Table