Conny Dekker

Identification of an unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene

Background 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 from these patients displayed a defect in the import of peroxisomal matrix and membrane proteins, resulting in a total absence of peroxisomal remnants. Objective To report on six 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 leucodystrophy 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 variables in fibroblasts were only mildly abnormal or within the normal range. Immunofluorescence microscopy 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. Conclusions 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, was identified. 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.

Genetic basis of peroxisome-assembly mutants of humans, Chinese hamster ovary cells, and yeast: identification of a new complementation group of peroxisome-biogenesis disorders apparently lacking peroxisomal-membrane ghosts.

We thank K. Hori for technical assistance, M. Ohara for helpful comments, and K. Kamijo for human PMP70 cDNA and an anti-ALDP antibody. This study was supported in part by a grant-in-aid for scientific research (08670870) from the Ministry of Education, Science, Sports, and Culture of Japan, by a research grant from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan, and by a research grant from Ono Medical Research Foundation.

Immunoblot analysis of peroxisomal proteins in liver and fibroblasts from patients

SummaryIdentification of a patient as suffering from a peroxisomal disorder usually starts by the finding of elevated very long-chain fatty acids in plasma and/or serum. This is followed by more detailed studies in blood, fibroblasts and tissues, including immunoblot analysis. Indeed, immunoblot analysis has become a valuable tool in the correct diagnosis and assignment of individual patients, except for X-linked adrenoleukodystrophy (X-ALD). We describe a simple immunoblotting procedure applicable to liver and fibroblast homo-genates using antibodies raised against catalase and the three β-oxidation enzyme proteins acyl-CoA oxidase I, bifunctional protein and peroxisomal thiolase. The same procedure can also be used for chorionic villus biopsy specimens and has now become the method of choice for the prenatal diagnosis of Zellweger syndrome (and other disorders of peroxisome biogenesis) and rhizomelic chondrodysplasia punctata.

Genotype-phenotype correlation in PEX5-deficient peroxisome biogenesis defective cell lines

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 (PBD) identified 11 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 (VLCFA) concentrations, peroxisomal β‐and α‐oxidation, dihydroxyacetone‐phosphate acyltransferase (DHAPAT) activity, peroxisomal thiolase, and catalase immunofluorescence. Mutation analysis of the PEX5 gene revealed 11 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. Hum Mutat 0,1–6, 2008.

Disorders of peroxisome biogenesis: Complementation analysis shows genetic heterogeneity with strong overrepresentation of one group (PEX1 deficiency)

The peroxisomal disorders represent a group of genetic diseases in man in which there is an impairment in one or more peroxisomal functions (Wanders et al 1995). One group of peroxisomal disorders involves the disorders of peroxisome biogenesis (PBDs), which include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD) plus a number of phenotypic variants not easily assignable to one of these entities. In addition to these disorders, rhizomelic chondrodysplasia punctata (RCDP) type 1 should also be included in the group of biogenesis disorders. In RCDP type I there is a selective defect in the import of a group of peroxisomal proteins with a peroxisome-targeting signal type 2 (PTS2) motif due to mutations in the PEX7 gene coding for the PTS2-receptor (Braverman et al 1997; Motley et al 1997; Purdue et al 1997). Earlier studies have already revealed genetic heterogeneity among patients affected by disorders of peroxisome biogenesis (Brul et al 1988; McGuiness et al 1990; Moser et al 1995; Poll-The et al 1989; Roscher et al 1989; Shimozawa et al 1993; Yajima et al 1992). We have now extended these studies to 113 patients by performing complementation analysis in fibroblasts using catalase immunofluorescence as a parameter for complementation.

Enzymatic diagnosis of Sjögren-Larsson syndrome using electrospray ionization mass spectrometry

BACKGROUND Sjögren-Larsson syndrome is a metabolic disorder characterized by accumulation of long-chain fatty alcohols in plasma of patients due to mutations in the ALDH3A2 gene, that codes for a microsomal fatty aldehyde dehydrogenase (FALDH). Recent studies have demonstrated that FALDH is involved in the last step of the conversion of 22-hydroxy-C22:0 into the dicarboxylic acid of C22:0 (C22:0-DCA). METHODS FALDH activity was determined by incubating fibroblast homogenates with omega-hydroxy-C22:0 in the presence of NAD(+). Electrospray ionization mass spectrometry (ESI-MS) was used to quantify the amounts of C22:0-DCA produced. RESULTS All SLS patients were deficient in C22:0-DCA productions with activities ranging from 3.2-26.3% of mean control. CONCLUSIONS The new assay described in this paper has substantial advantages over previous assays, and allows for the easy, reliable and rapid diagnosis of SLS.

X-linked adrenoleukodystrophy: Improved prenatal diagnosis using both biochemical and immunological methods

adrenoleukodystrophy (X-ALD; McKusick 300100) is one of the more X-Linked frequent peroxisomal disorders with a variable phenotypic expression et al (Moser The most notorious variant is childhood cerebral ALD (CCALD) occurring 1995). in previously healthy boys before the age of 10 years. Initial symptoms frequently include behavioural abnormalities and loss of school performance associated with visual and/or auditory disturbances and abnormal gait. The course of the disease is progressive, culminating within a few years in dementia, blindness, quadriplegia and death. Adrenomyeloneuropathy is the second most commonly observed phenotype of ALD and presents in adulthood with primary involvement of spinal cord and peripheral nerves. Biochemically, all ALD phenotypes in the hemizygous form show elevated plasma levels of very long-chain fatty acids (VLCFA) reÑected in increased values for and The X-ALD gene has recently been 26>0 , 24>022>0 26>022>0 . identiÐed et al and codes for a peroxisomal membrane protein (Mosser 1993) involved in the transport of VLCFA across the peroxisomal membrane. In recent years many di†erent mutations in the X-ALD gene have been described et al (Moser 1995). The availability of antibodies raised against di†erent portions of the X-ALD protein has allowed studies at the protein level either by immunoblotting or/and by immunoÑuorescence et al et al et al (Watkins 1995 ; Feigenbaum 1996 ; Kemp 1996). In the majority of patients the ALD-protein (ALDP) was found to be fully absent, suggesting that ALDP analysis in chorionic villus cells or amniocytes may well be very useful for prenatal diagnosis, especially since reports have been published showing that analysis of VLCFA alone may lead to erroneous conclusions et (Gray al 1995).