HUMAN MUTATION Mutation in Brief #933 (2006) Online

MUTATION IN BRIEF

Identification of Novel Mutations in PEX2, PEX6, PEX10, PEX12, and PEX13 in Zellweger Spectrum Patients Cindy Krause+, Hendrik Rosewich+, Melissa Thanos, and Jutta Gärtner*

Department of Pediatrics and Pediatric Neurology, Georg August University, Göttingen, Germany

+ These authors contributed equally to this work.

*Correspondence to: Jutta Gärtner, M.D., Dept. of Pediatrics and Pediatric Neurology, Georg August University, Faculty of Medicine, Robert-Koch-Str. 40, 37075 Göttingen, Germany; Tel: +49-551-398035; E-mail: [email protected]

Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Ga 354/5-1 and 5-2.

Communicated by Ronald J.A. Wanders

Mutations in each of the 13 identified human PEX genes are known to cause a peroxisomal biogenesis defect (PBD). Affected patients can be divided into two broad clinical spectra: the Zellweger spectrum, which accounts for about 80% of PBD patients, and the rhizomelia chondrodysplasia punctata (RCDP) spectrum. The clinical continuum of Zellweger spectrum patients extends from Zellweger syndrome (ZS) as the prototype and the most severe entity of this group to neonatal adrenoleukodystrophy (NALD) as an intermediate form and infantile Refsum (IRD) disease as the mildest variant. Characteristic features of ZS patients are dysmorphic features, severe neurological impairment, liver dysfunction, and eye and skeletal abnormalities. Similar but less severe clinical signs are seen in patients with NALD and IRD. In this study ten clinically and/or biochemically well-characterized patients with classical ZS were investigated for defects in all known human PEX genes. We identified two novel mutations in PEX2 (official symbol, PXMP3), two novel mutations in PEX6, two novel mutations in PEX10, one novel mutation in PEX12, and one novel mutation in PEX13. © 2006 Wiley-Liss, Inc.

KEY WORDS: Zellweger syndrome spectrum; PEX2; PEX6; PEX10; PEX12; PEX13; PXMP3

INTRODUCTION Peroxisomes are ubiquitous components of eukaryotic cells. Their enzymes contribute to multiple metabolic processes, most of which involve the metabolism of lipids (Wanders, 2004). Mutations in genes encoding peroxisomal metabolic enzymes cause various human diseases. The most dramatic loss of peroxisome function is observed in peroxisome biogenesis disorders (PBDs; MIM# 601539). In these patients synthesis of peroxisomal proteins is normal, but peroxisomal membrane biosynthesis or import of peroxisomal enzymes into the lumen is impaired. Peroxisomal proteins are encoded by nuclear genes, synthesized on free cytosolic ribosomes and imported post-translationally. Matrix proteins are then targeted to the peroxisome using two different peroxisomal targeting signals (PTS1/PTS2). Membrane proteins have different and distinct targeting signals. Conceptually, the

Received 20 January 2006; accepted revised manuscript 6 July 2006.

© 2006 WILEY-LISS, INC. DOI: 10.1002/humu.9462

2 Krause et al.

PBDs can be caused by defects in any of several processes, including the synthesis of peroxisomal membranes, the recognition of newly synthesized peroxisomal proteins or any of the downstream steps in their import. Although all PBD patients show some defects in peroxisomal matrix protein import, they display a great genetic and phenotypic heterogeneity. In early studies three overlapping clinical entities were recognized: ZS (MIM# 214100), NALD (MIM# 202370), IRD (MIM# 266510) and RCDP (MIM# 215100), which is characterized by distinct phenotypes such as unique skeletal abnormalities, a more limited set of peroxisomal metabolic defects and a PTS2 specific protein import defect. ZS patients display dysmorphic features such as hypertelorism, epicanthus, broad nasal bridge and large fontanelles with wide sutures. They also suffer from severe neurological dysfunction, as well as hepatic and renal defects, and rarely survive their first year. Similar but less severe phenotypes are observed in the patients with NALD, who survive up to a decade. IRD patients are even more mildly affected and some survive beyond their third decade. Genes involved in peroxisome biogenesis are defined as PEX genes encoding peroxins. Two strategies have been proven to be effective in identifying responsible PEX genes for the PBDs: homology probing and functional complementation of peroxisome-deficient Chinese hamster ovary cells with mammalian cDNA expression libraries. Numerous PEX genes and peroxins required for peroxisome biogenesis were identified in unicellular eukaryotes like yeast. Subsequent computer-based searches of mammalian databases with these yeast peroxin sequences identified many of their human homologs. Screening these homologs for involvement in the PBDs by mutation analysis and functional complementation assays in fibroblasts of PBD-patients led first to the identification of PEX5 (PXR1; MIM# 600414) as the gene defective in the PBD complementation group 2 (CG2) (Dodt, et al., 1995). Successive use of this method identified genes defective in PBD CG1 (PEX1; MIM# 602136) (Portsteffen, et al., 1997; Reuber, et al., 1997), CG3 (PEX12; MIM# 601758) (Chang, et al., 1997), CG4 (PEX6; MIM# 601498) (Yahraus, et al., 1996), CG7 (PEX10; MIM# 602859) (Warren, et al., 1998), CG9 (PEX16; MIM# 603360) (Honsho, et al., 1998), CG11 (PEX7; MIM# 601757) (Braverman, et al., 1997; Motley, et al., 1997), CG12 (PEX3; MIM# 603164) (South, et al., 2000) and CG13 (PEX13; MIM# 601789) (Liu, et al., 1999). Functional complementation of peroxisome-deficient Chinese hamster ovary cells with mammalian cDNA expression libraries revealed the primary genetic defect of CG10 (PEX2; official symbol, PXMP3, MIM# 170993) (Shimozawa, et al., 1992). Successful elaboration of this strategy led to the independent identification of the genes defective in CG3 (PEX12) (Okumoto, et al., 1998), CG4 (PEX6) (Fukuda, et al., 1996), CG14 (PEX19; official symbol, PXF, MIM# 600279) (Matsuzono, et al., 1999) and CG8 (PEX26; MIM# 608666) (Ghaedi, et al., 1999). So far, the primary genetic defect of all 13 CGs is known. Although PEX1 mutations contribute for more than 60% of sequence alterations in PBD patients (Rosewich, et al., 2005), there is an expanding proportion in PBD patients revealing mutations in other PEX genes (Steinberg, et al., 2004). Here we describe eight novel mutations in five different PEX genes. New insertion and duplication mutations were detected in PEX6, whose mutations contribute for about 11% of all PBD sequence alterations. Identification of diverse mutations in PEX genes in this study suggests complexity of genetic heterogeneity in the PBD patients. A database comprising all known sequence alterations in PEX genes will support more efficient genetic counselling and prenatal diagnosis for the parents. Moreover, such databases will be the fundament for basic researchers to understand the biogenesis of peroxisomes through functional studies in cell and animal models.

PATIENTS, MATERIAL AND METHODS Patient Cell Lines We screened 40 individual patient skin fibroblast cell lines from 38 different families of Turkish, Romanian and German origins that were diagnosed with ZS. Patients PBD-HR1, -HR2, -HR3, -HR7 and -HR11 have consanguineous parents; consanguinity could be excluded for all other patients, except for patient PDB-HR10. The cell lines were cultured in Dulbecco's Modified Eagle Medium (low glucose) (PAA Laboratories GmbH, Cölbe, Germany, http://www.paa.at/) supplemented with 10% fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany, http://www.biochrom.de/), 100mg/ml penicillin and 100U/ml streptomycin (PAA Laboratories GmbH).

Transfection In order to determine which of the known PEX genes may induce the peroxisome biogenesis disorder, the patient fibroblast cells were transfected with pcDNA3 plasmids (Invitrogen, Invitrogen GmbH, Karlsruhe, Germany, http://www.invitrogen.com) containing each PEX genes: PEX1 (Portsteffen, et al., 1997; Reuber, et al., Novel PEX Gene Mutations 3

1997), PEX3 (South, et al., 2000), PEX5 (Dodt, et al., 1995), PEX6 (Yahraus, et al., 1996), PEX10 (Warren, et al., 1998), PEX12 (Chang, et al., 1997), PEX13 (Bjorkman, et al., 1998), PEX19 (Sacksteder, et al., 2000) or PEX26 (Weller, et al., 2005). Full-length ORF (open reading frame) was amplified from cDNA clones IRAU0969E0910D6 (PEX2), IRAUp969G0833D6 (PEX14) and IRAUp969D0218D6 (PEX16) and cloned into pcDNA3 to generate expression constructs for transfection. Detailed cloning schemes are available on request. Each transfection reaction contained 10µl Effectene, 3.2µl Enhancer and 0.4µg DNA and was performed following the instruction by the manufacturer (Qiagen, Qiagen GmbH, Hilden, Germany, http://www1.qiagen.com). EGFP- Peroxi (Clontech, Mountain View, CA, USA, http://www.clontech.com) containing PTS1 was cotransfected to examine import of PTS1-proteins in the patient cells. After 48 hours, the cells were analyzed under fluorescence microscope (Nikon GmbH, Düsseldorf, Germany, http://www.nikon.de) to determine whether they reveal punctate peroxisomal or diffuse cytosolic localization of EGFP-Peroxi.

RNA and Genomic DNA Isolation and cDNA Synthesis Following the manufacturer's instruction, RNeasy Mini Kit (Qiagen) was used to extract total RNA from patient fibroblast cells that demonstrated unambiguous complementation by single PEX gene. Then, SuperScript III First- Strand Synthesis System for RT-PCR (Invitrogen) was used to synthesize cDNA from 2µg RNA. Complete ORF of respective PEX transcripts was amplified and sequenced on an ABI PRISM 3100 Avant (Applied Biosystems, Darmstadt, Germany, http://www.appliedbiosystems.com). The sequences were compared to the following reference sequences, respectively: PEX2 (NM_000318.1), PEX6 (NM_000287.2), PEX10 (NM_002617.3), PEX12 (NM_000286.1) and PEX13 (NM_002618.2). All mutations were verified in the genomic DNA, which was extracted from the patient fibroblast cells using DNeasy Tissue Kit (Qiagen). All identified mutations were confirmed by direct sequencing of two individual PCR amplification products on forward and reverse strands. 63 control individuals (126 control alleles) were analysed for the absence of p.Trp313Gly missense mutation in PEX13. Primer sequences as well as PCR and sequencing conditions are available on request. The mutation numbering is based on cDNA sequence and +1 corresponds to the A of the ATG translation initiation codon in the reference sequence. The mutation nomenclature follows the guidelines recommended on the Mutation Nomenclature Homepage at the HGVS website (http://www.hgvs.org/mutnomen/).

RESULTS AND DISCUSSION We complemented skin fibroblast cell lines from 40 Zellweger spectrum patients with PEX expression constructs in order to identify the putative disease causing PEX genes. 28 Patients had a PEX1 defect and one patient had the most common PEX10 defect c.884_885delCT, pL272fs (Shimozawa, et al., 2003). Here we report eight novel mutations found in five known PEX genes within the ten individuals (Table 1). We identified a nonsense as well as a frameshift mutation in PEX2 (p.Gln39X, p.Phe278LeufsX3), two different frameshift mutations in PEX6 (p.Ser232HisfsX15, p.Gly473ArgfsX13), two nonsense mutations in PEX10 (p.Arg244X, p.Trp294X), a nonsense mutation in PEX12 (p.Ser292X) and a missense mutation in PEX13 (p.Trp313Gly). PEX2 gene was the first gene identified to be mutated in Zellweger spectrum patients and eight homozygous mutations have been found so far (Gootjes, et al., 2004; Shimozawa, et al., 1993; Shimozawa, et al., 1992; Steinberg, et al., 2004). PEX2 is an integral membrane protein with two transmembrane domains (transmembrane domain 1 (TMD1): amino acid (AA)140-159, TMD2: AA195-213) and a RING finger motif (AA144-283) at the C-terminal end (Gootjes, et al., 2004). c.115C>T mutation in patient PBD-HR1 leads to the introduction of a termination codon at Gln39, which generates a truncated protein lacking both transmembrane domains. Most likely, this protein is unable to localize correctly to the peroxisomal membrane and thus impairs peroxisomal biogenesis. The severe clinical phenotype and the early death at the age of two months of patient PBD-HR1 suggests absence of functional PEX2 protein. Patients PBD-HR2 and PBD-HR3, who are siblings, contain the PEX2 mutation c.834-838delTACTT. The encoded protein shows a frameshift within the conserved RING finger motif. Previous study revealed that a patient containing truncated PEX2 protein, which lacks a complete RING finger motif, is clinically only mildly affected (Shimozawa, et al., 2000). However, our patients PBD-HR2 and PBD-HR3, who lack only the last six amino acids of this RING motif, are rather severely affected as they died at the age of three and four months, respectively. Possibly, the mutated PEX2 fails to interact with other proteins and thereby disturbs the peroxisomal import mechanism. The unrelated patients PBD-HR4 and PBD-HR5share the same mutation in PEX6 gene (p.Ser232HisfsX15) and showed comparable clinical phenotypes. PEX6 protein is a member of the AAA ATPase family and contains two 4 Krause et al.

AAA cassettes (Collins and Gould, 1999; Zhang, et al., 1999) that are highly conserved between human and rat (Fukuda, et al., 1996). Especially, the second AAA cassette is essential for PEX6 protein function in import of peroxisomal matrix proteins since a missense mutation eliminated its biological activity (Yahraus, et al., 1996). Both frameshift mutations identified (p.Ser232HisfsX15; p.Gly473ArgfsX13) introduce early termination codons and thereby generate PEX6 proteins lacking the second AAA cassette. The early age at death of less than four months supports a major lack of PEX6 function in the patients PBD-HR4, -HR5 and -HR6. Previous studies revealed that PEX10 protein contains a C-terminal RING finger domain (AA273-311) which is functionally essential (Warren, et al., 1998; Warren, et al., 2000). Sequencing of both cDNA and genomic DNA revealed that patient PBD-HR7 contains homozygous p.Arg244X mutation whereas patient PBD-HR8 carries heterozygous p.Arg244X and p.Trp294X mutations. Notably, we could detect only p.Trp294X mutation in cDNA of patient PBD-HR8. This result suggests that mRNA leading to p.Arg244X mutation may be unstable due to the early nonsense codon and may not be expressed properly. Both mutations generate a putative PEX10 protein lacking either complete or part of the zinc-binding RING finger domain. Earlier findings demonstrated that fibroblasts of a ZS patient cannot be complemented for peroxisome biogenesis when they were transfected with a mutagenized PEX10 cDNA containing L272fs (Shimozawa, et al., 2003). This result suggests that L272fs, which truncates most of the RING finger, obliterates biological activity of PEX10. Similarly, Arg244X and Trp294X mutation may lead to the loss of PEX10 function. The very short survival age of four days for patient PBD-HR7 further supports severely impaired function of PEX10. PEX12 encodes an integral peroxisomal membrane protein that spans the peroxisome membrane twice and contains a zinc-binding RING domain (AA260-359, (Chang, et al., 1999)) at its C-terminus (Okumoto and Fujiki, 1997). Mutations, which truncate upstream of the cytoplasmically exposed zinc-binding RING domain, were shown to severely limit PEX12 activity (Chang, et al., 1999). Mutation in patient PBD-HR10 deletes part of the zinc-binding RING domain, which is required for the interaction of PEX12 with PEX5 and PEX10 (Chang, et al., 1999). Further studies may reveal whether this partial truncation would affect the interaction between PEX12 and PEX5 as well as PEX10. Whereas numerous mutations have been identified in PEX2, PEX6, PEX10, and PEX12, only two mutations have been described so far for PEX13. Here we report a novel missense mutation p.Trp313Gly in PEX13, which encodes an integral peroxisomal membrane protein. Absence of this missense mutation in 126 control alleles indicates that it is most likely a disease causing mutation rather than a nonpathogenic polymorphism. In yeast Pex13p functions as a docking factor for PTS1 receptor Pex5p (Gould, et al., 1996) and also interacts with Pex14p via its SH3 domain at C-terminal (Barnett, et al., 2000). The mutated Trp313 in patient PBD-HR11 lies within N- Src loop, which contributes significantly to ligand recognition and specificity by the SH3 domain (Lee, et al., 1995; Wu, et al., 1995). Earlier studies demonstrated that the tryptophan residue plays a key role in the direct recognition of the P-X-X-P ligand backbone, which can also be found in Pex14p sequence (Barnett, et al., 2000). Alignments of PEX13 protein sequence with all known peroxin13 sequences in various species revealed that Trp313 is absolutely conserved in all sequences (Fig. 1). When Trp349 in yeast, which corresponds to Trp313 in human, was substituted by alanine, Pex13p could not interact with Pex14p anymore (Barnett, et al., 2000). This result suggests strongly that this tryptophan residue is essential for the specific interaction between Pex13p and Pex14p. Similarly, the substitution of Trp313 by Gly in human PEX13 may eliminate the interaction between PEX13 and PEX14. In summary, we observe that the genotype correlates well with phenotype of ZS patients. Nonsense mutations, which generate short fragments of PEX proteins or deletions of essential functional domains, appear to induce severe clinical phenotypes, whereas missense mutations lead to milder clinical phenotypes. Our results support further the hypothesis that variations in the clinical as well as cellular phenotypes are based on the differences in allele severity. Novel PEX Gene Mutations 5

Table 1. Clinical Data and PEX-Gene Mutations in PBD Patients Age at Phenotype/ Age at Patient Dysmorphic Clinical last Affected Mutation nucleotide Mutation protein Ethnic Sex Involved death No. features2 course3 exam gene level level background systems1 (mo.) (mo.) PBD- C (PS, GF, H), LF/WS, BN, c.115C>T F RP 2 2 PEX2 p.Gln39X Turkish HR1 H (H, RC) E, EE c.115C>T

C (PS, GF, H, PBD- c.834_838delTACTT M S), O (C), H LF/WS, SF RP 3 PEX2 p.Phe278LeufsX3 Turkish HR2 c.834_838delTACTT (H, ELE) C (PS, GF, H, PBD- c.834_838delTACTT F S), O (N), H ? RP 4 PEX2 p.Phe278LeufsX3 Turkish HR3 c.834_838delTACTT (ELE)

PBD- C (H, SPR), O LF/WS, HF, c.685_686insAG F RP 3 3 PEX6 p.Ser232HisfsX15 German HR4 (N), H (ELE) BN, H, SF c.685_686insAG

C (PS, GF, H), PBD- LF/WS, HF, 2 2 c.685_686insAG M H (H, ELE, RP PEX6 p.Ser232HisfsX15 Romanian HR5 BN, EE, SF weeks weeks c.685_686insAG RC), S (CS)

C (PS, GF, H, PBD- LF/WS, HF, c.1415dupC F S), H (H, RP 3.5 PEX6 p.Gly473ArgfsX13 Turkish HR6 BN c.1415dupC ELE), O (C)

PBD- C (H, S), H c.730C>T M BN, EE, SF RP 4 days 4 days PEX10 p.Arg244X Turkish HR7 (RC) c.730C>T

PBD- c.730C>T p.Arg244X F ? ? ? ? ? PEX10 German HR8 c.881G>A4 p.Trp294X C (PS, GF, H, PBD- LF/WS, HF,E, c.875_876delCT F SPR, S), H (H, RP 3 3 PEX12 p.Ser292X Turkish HR10 EE, SF c.875_876delCT ELE) O (N) caudal PBD- neuropore, c.937T>G F C (H), O (C) P 31 31 PEX13 p.Trp313Gly Turkish HR11 inverted c.937T>G nipples 1: Phenotype/Involved systems: C: Cerebral (PS: poor sucking, GF: gavage feeding, H: hypotonia, SPR: severe psychomotor retardation, S: seizures) 1: Phenotype/Involved systems: O: Ocular (C: cataract, RP: retinitis pigmentosa, OA: optic atrophy, N: nystagmus) 1: Phenotype/Involved systems: H: Hepatorenal (H: Hepatomegaly, LF: liver fibrosis, ELE: elevated liver enzymes, RC: renal cysts) 1: Phenotype/Involved systems: S: Skeletal systems (CS: calcific stipling) 2: Dysmorphic features: LF/WS: large fontanelle/wide sutures, HF: high forehead, BN: broad nasal bridge, H: hypertelorism, E: Epicanthus, EE: external ear deformity, SF: sickle foot. 3: Clinical course: S: stable, P: progressive, RP: rapidly progressive 4: Although we could detect two heterozygous mutations in the genomic DNA of the patient PBD-HR8, we could detect only c.881G>A mutation in the cDNA. Reference sequences: PEX2 (NM_000318.1), PEX6 (NM_000287.2), PEX10 (NM_002617.3), PEX12 (NM_000286.1), and PEX13 (NM_002618.2). The mutation numbering is based on cDNA sequence and +1 corresponds to the A of the ATG translation initiation codon in the reference sequence. 6 Krause et al.

HsPex13 283FAAV----SEEEISFRAGDMLNLALKEQQPK--VRGW LLAS-LDGQTTGL CgPex13 FNAV----SDEEISFRAGDMLNLALKEQQPK--VRGW LLAS-LDGQTTGL MmPex13 FVAV----SDEEISFRAGDMLNLALKEQQPK--VRGW LLAS-LDGQTTGL CePex13 FQAS----NEQELSFMNGETLRVAPKEEQPR--VRGW LLASVADGSRIGL ScPex13 FVPE---NPEMEVALKKGDLMAILSKKDPLG-RDSDW WKVRTKNG-NIGY PpPex13 FNPE---NEEMELKLARGELMAILSKTEPNSNQESTW WKCRSRDG-KVGF AfPex13 YTPESQESAGIDLAVKKGDIVAVLSKTDPMG-NASEW WRCRARDG-RVGY CnPex13 FTP----TEEWELGLGRDEIVAVLEKRGEG---MNSW WRGRTRDG-RTGW CaPex13 FNPQ---NPQVEAPLEPKEIVAILDSRD------NW LRIRKRSG-TMGW : . : . : : : . * .* *

Figure 1. Trp313 in the SH3 domain of HsPEX13 protein is evolutionarily conserved. SH3 domains of human Pex13 (HsPex13; Q92968), Chinese hamster Pex13 (CgPex13; Q9QYL1), mouse Pex13 (MmPex13; Q9EPK1), putative Caenorhabditis elegans Pex13 (CePex13; Q19951), Saccharomyces cerevisiae Pex13 (ScPex13; P80667), Pichia pastoris Pex13 (PpPex13; Q92266), putative Aspergillus fumigatus (AfPex13; Q4WCA2), putative Cryptococcus neoformans Pex13 (CnPex13; Q5KKC0), and putative Candida albicans (CaPex13; Q5A425) were aligned using ClustalW. Trp314 (black box), which is mutated to glycine in patient PBD-HR11, is absolutely conserved among all the species.

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