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