Petra A. W. Mooijer

Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis

In the past few years, many patients have been described who have a defect of unknown origin in the peroxisomal beta-oxidation pathway. Complementation analysis has been done by various groups to establish the extent of the genetic heterogeneity among the patients. These studies were based on the use of two established cell lines, one with a deficiency of acyl-CoA oxidase and one with a deficiency of l-bifunctional protein (l-BP), and they showed that most patients belong to the l-BP-deficient group. However, molecular analysis of the cDNA encoding l-BP in patients failed to show any mutations. The recent identification of a new d-specific bifunctional protein (d-BP) prompted us to reinvestigate the original patient with presumed l-BP deficiency. In a collaborative effort, we have now found that the true defect in this patient is at the level of the d-BP and not at the level of the l-BP. Our results suggest that most, if not all, patients whose condition has been diagnosed as l-BP are, in fact, d-BP deficient. We tested this hypothesis in nine patients whose condition was diagnosed as l-BP deficiency on the basis of complementation analysis and found clear-cut mutations in the d-BP cDNA from all patients.

Genetic classification and mutational spectrum of more than 600 patients with a Zellweger syndrome spectrum disorder.

The autosomal recessive Zellweger syndrome spectrum (ZSS) disorders comprise a main subgroup of the peroxisome biogenesis disorders and can be caused by mutations in any of 12 different currently identified PEX genes resulting in severe multisystemic disorders. 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. These 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. In a majority of the cell lines we subsequently determined the underlying mutations by sequence analysis of the implicated PEX genes. 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. Hum Mutat 31:1–11, 2010.

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.

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.

D-Hydroxyacyl-CoA Dehydrogenase Deficiency

The second and third steps of peroxisomal β-oxidation are catalysed by two multifunctional enzymes: D-bifunctional protein and L-bifunctional protein. Here we show that fibroblasts of a patient described as being deficient in the 3-hydroxyacyl-CoA dehydrogenase component of D-bifunctional protein and fibroblasts of a patient described as being deficient in L-bifunctional protein do not complement one another. Using a newly developed method to measure the activity of D-bifunctional protein in fibroblast homogenates, we found that the activity of the D-bifunctional protein was completely deficient in the patient with presumed L-bifunctional protein deficiency.